Image analysis was used to study the cytokine-inhibitory effect of the nitric oxide inhibitor tetravalent guanylhydrazone (CNI-1493) in individual immunocytochemically stained human peripheral blood mononuclear cells (PBMC). CNI-1493 inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-1beta, IL-6, and IL-8 production whether or not LPS stimulation was enhanced by interferon (IFN)-gamma priming. Addition of TNF-alpha to CNI-1493-exposed LPS-stimulated cells partially restored the incidence of IL-1alpha-, IL-1beta-, and IL-8-producing cells. TNF-alpha production induced by costimulation by ligation of CD3 and CD28 was inhibited by CNI-1493 in monocytes but not in T lymphocytes. The prevalence of IL-2-, IFN-gamma-, and TNF-beta-producing T cells was not reduced by CNI-1493. Phorbol ester and ionomycin activation also resulted in a CNI-1493 -induced inhibition of TNF-alpha in monocytes but resistant production of TNF-alpha, IL-2, and IFN-gamma by T cells. Thus, CNI-1493 preferentially inhibited synthesis of proinflammatory cytokines in monocytes.