The activity (carbon clearance) of the reticuloendothelial system (RES) of mice inoculated intraperitoneally with N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide, MDP) was greatly stimulated 1 day, but not 7 days after MDP treatment. No enhancement of resistance to ectromelia virus infection and influenza virus infection in mice treated with MDP was observed. In mice splenectomized 1 week after MDP pretreatment, normal levels of circulating interferon were produced in response to Newcastle disease virus (NDV), whereas in the mice treated with MDP after splenectomy, circulating interferon levels were reduced to the same level as produced in the MDP-untreated and splenectomized mice. Interferon production in response to NDV was augmented in non-adherent peritoneal and spleen cell cultures derived from MDP-pretreated mice, whereas it was reduced in peritoneal and splenic macrophage cultures. These results suggest that the non-adherent spleen cells activated with MDP were disseminated from the spleen to other organs, that the lack of enhancement of interferon production in mice pretreated with MDP might be due to reduced interferon production in macrophages, and that the activation of the RES of the whole body by MDP did not correlate with the enhancement of interferon production in spleen cells or with the reduction of interferon production in macrophages.