In The Sisters Brothers, Eli and Charlie are both contract killers in the Old West, a stereotypically masculine occupation. However, while Charlie lives up to this stereotype at the beginning of the novel, Eli subverts it with his sentimentality and reflectiveness. Even Eli's role as narrator means that he cannot fulfil the traditional "strong and silent" persona adopted by Charlie, at least as far as the reader is concerned. Eli's neuroticism as he wonders how much to tip a clerk in a hotel or his simple pleasure in acquiring a toothbrush are at odds with the grim and toxic masculinity expected of the hired killer.
At the beginning of the novel, Eli and Charlie look after each other in different ways. Charlie takes on the more traditionally masculine role, leading the expedition and handling most of the violence, while Eli must nurse Charlie through the sickness caused by his drinking. Eli continues in this feminine role when Charlie's hand is burned and amputated.
However, the loss of his shooting hand also robs Charlie of his masculinity, and he becomes a more pitiable, childlike figure. It is therefore Eli who has to take responsibility for killing the man who hired them and ending their murderous career (albeit, ironically, with a murder). By the end of the novel, the contrast between the two is no longer so marked, and this increased similarity emphasizes their brotherhood.