Deer mice (Peromyscus maniculatus) are the principal reservoir hosts of Sin Nombre hantavirus (SNV). Once infected, deer mice remain persistently infected for the remainder of their lives. It is unknown what immune evasion strategies SNV uses to elude the host immune response, but evidence suggests that macrophages may be one target of viral infection. If so, SNV may interfere with normal macrophage functions that impair the immune response and contribute to evasion. Previous attempts to isolate and culture deer mouse macrophages have not been successful. We report here the first successful isolation and activation of deer mouse peritoneal macrophages. Isolated cells exhibited macrophage morphology and expressed several macrophage cytokine and chemokine genes when stimulated with lipopolysaccharide, including tumor necrosis factor, macrophage inflammatory protein-1α and interleukin-12. The ability to culture deer mouse macrophages will permit examination of SNV infectivity and consequences in macrophage functions.