Splenic macrophage tumoricidal activity was examined and a splenic macrophage tumoricidal assay was established. Initially, mixtures of lipopolysaccharide (LPS) and spleen single cell suspensions (SSCS) were cultured for 1-4 days. Adherent macrophages, washed free of nonadherent cells and LPS, were then examined and were found to lack tumoricidal activity in a standard 18-h 51Cr release assay. However, tumoricidal activity was generated if LPS was added to the SSCS cultures at later time points during the 4-day incubation period; maximal activity was seen when LPS was added on day 3. In parallel, significant changes in macrophage autofluorescence and morphology, but not phenotype, were observed. Next, SSCS were cultured for 1-4 days without stimulating agents. Adherent macrophages were then washed free of nonadherent cells and LPS was added. Significant tumoricidal activity developed in time- and LPS concentration-dependent fashions. The presence of nonadherent spleen cells in physical contact with the macrophages during the SSCS culture was essential for the macrophages in the resultant monolayer to be responsive to LPS. Activated splenic macrophage-mediated lysis of tumor cells was shown to depend on the contact between the two cells.