Bone marrow-derived murine macrophages were used to study the relationship between the proliferative response of macrophages to macrophage colony-stimulating factor (CSF-1) and their activation for cytocidal activity against tumour cells. Macrophage activation required two sequential signals. Lymphokines (gamma interferon, interleukin-4) provided the first (priming) signal; bacterial products (lipopolysaccharide, lipophilic muramyl tripeptide, lipopeptide 31362, pertussis toxin) provided the second (triggering) signal. Both priming and triggering agents inhibited [3H]-thymidine uptake by macrophages stimulated with CSF-1. The antiproliferative activity of priming and triggering stimuli was synergistic. Pretreatment with triggering stimuli at 37 degrees C caused a rapid reduction of the subsequent binding of [125I]-CSF-1 to the cell surface at 4 degrees C, whereas priming agents had relatively little effect. Growth inhibition by both priming and triggering agents was largely reversible by washing the cells, and occurred even when they were added as long as 24 h after the growth factor. The ability of pertussis toxin to both inhibit CSF-1-induced proliferation and trigger cytotoxicity in macrophages suggests the involvement of a regulatory GTP-binding protein (G protein) in both processes.