Sublethal infection of mice with Listeria monocytogenes was accompanied by an influx of immigrant macrophages into the liver and the generation of substantial H2O2 by isolated liver macrophages. H2O2 production paralleled the course of infection and, after resolution of granulomata, returned to the low levels seen in normal livers. To assess the activation status of Kupffer cells and immigrant macrophages in listeriosis, a separation protocol was developed based on the differential adherence properties of the two macrophage populations. As in the steady state, Kupffer cells in listeriosis failed to generate significant levels of H2O2 and did support the replication of internalized toxoplasmas. Immigrant macrophages produced substantial levels of H2O2 and could quantitatively account for H2O2 production by total liver macrophages. Our findings suggest distinct functions for Kupffer cells and immigrant macrophages.