Cal's burgeoning awareness of his difference propels the book forward. He holds himself back emotionally because of the weight of being both male and female, which compels him to keep his distance. Cal is a nearly omniscient narrator; he admits to making up what he can't know and knowing things he couldn't possibly know. The conceit of Middlesex is that Cal is writing it in part to understand himself. The novel jumps around in time and tense, imitating the style of a storyteller who has many asides.
Desdemona, a country girl who husbands her family's silkworm cocoonery, loses her parents in Greek-Turkish violence. Grief-stricken, afraid, and longing for closeness, she agrees to marry her brother. Desdemona knows she has done something wrong. When learning she has a grandson rather than the granddaughter she watched grow up, Desdemona feels that all her secrets must come out. She remembers stories that her mother told her about girls changing into boys at puberty. Cal's condition is the result of his grandparents and parents sharing the same blood, as they were descendants of the same ancestor. After the death of her husband, Desdemona takes to her bed. She is a "sick person, imprisoned in a healthy body."
Lefty, felled by strokes, is silent throughout the latter part of the book. He is a gambler and has always been attracted by games of chance. Unlike his wife, he doesn't appear to have terrible pangs of guilt over their union. By marrying his sister, he ensures the transmission of the mutated chromosome. Lefty works hard, enjoys hashish, and explores the city of Detroit.
Milton grows increasingly anxiety-ridden and angry as the narrative moves along. He is used to solving problems in business and is willing to solve Callie's problem through hormone injections and surgery. There is no doubt that he loves his daughter; he feels fiercely protective of her. Milton is the most thoroughly assimilated member of his generation; he is a navy officer, a business man, a fierce champion of America and her policies abroad, even if they run counter to Greek interests. Milton is extorted for money by Father Mike, from whom he stole Tessie's heart. His sense that he knows best ends fatally, when he crashes his car while chasing Father Mike.
Tessie is desperate to understand her daughter. She is hyperaware when Dr. Luce says "your child" instead of "your daughter" and relieved when he begins to say "daughter" again. However, she understands, intuitively, that Callie isn't a girl. Yet Cal remains her daughter even as he becomes her son.
The Obscure Object is the object of Callie's first romantic obsession. While heterosexual girls develop crushes on other girls at her girls' school, Callie knows that her crush is different. Her "crocus" (genitalia) responds to the Object differently than to other girls. The Object trusts Callie, they become close friends, and they begin a sexual relationship. It is discovery of this relationship that causes Callie to flee into the farmer's field and be hit by a tractor. She never sees the Object again.
Father Mike, simmering for years after his fiancé Tessie marries Milton Stephanides, extorts money from Milton, professing to know where Callie is. His mild manner belies his anger and frustration; his wife barely conceals her contempt, he never becomes a head priest, and his family is poor compared to the Stephanides. It is he who leads Milton on the chase that leads to Milton's death.