Macrophage function depends on their in situ location. To test this hypothesis, we examined functional changes of the C2D macrophage cell line after adoptive transfer. In vitro, C2D macrophages reside early in the macrophage lineage and show little functional activity. After in vivo i.p. culture, C2D macrophage cells switch their cytokine/chemokine profile from primarily Th2 cytokines produced in vitro to a Th1 profile including MIP-1alpha, IL-6, and TNF-alpha. The in vivo environment also caused C2D macrophage cells to become more phagocytic than their in vitro counterparts. These data indicate that C2D macrophage cells exhibit distinct functions because of in vivo signals that are absent during in vitro culture.