Steinbeck's short novel follows a distinctly cyclical pattern, reinforced by the novel's use of setting. Opening in a glen by the Salinas River, the novel also ends in this idyllic place.
There is some significance to this natural setting as it is aligned with both freedom and innocence. Here George and Lennie are not subject to the rules of the ranch or the whims of the boss. They are at leisure. This natural place brings comfort.
Also, Lennie suggests that if George wanted to get rid of him, Lennie could run off further into nature. This comment renders the glen by the river as a passage and as a place where the relationship between the two men might end. Lennie speaks, in part, in innocence and, in part, with guile, as he attempts to manipulate his friend. More importantly, Lennie's connection to the natural world is emphasized by his threat to run away and "live in a cave".
There are only three other settings in the novel - the bunk house, the stable buck's room, and the barn.
The bunk house and the stable buck's room suggest the itinerant aspect of the lives of these men as it is an impersonal and definitively temporary living quarters. The stable buck's room suggests the isolation that Crooks feels, made to live alone, and this isolation resonates with the conversation Lennie and George have:
"Guys like us," George says, "that work on the ranches, are the loneliest guys in the world. They got no family. They don't belong noplace."
Curley's wife, Candy, and Crooks each connect with this isolation, symbolized by Crook's living quarters.
The barn, where Lennie kills Curley's wife, is a place where animals live and Lennie is drawn there. As a sign of domestication, the barn serves as a comment on Lennie's inability to truly adopt social rules and norms. He is more like animal than man in some ways (a notion that is clearly developed in the text through direct descriptions of Lennie resembling a bear and also a horse).