This study was initiated to investigate the possible involvement of prostaglandin H synthase in the staurosporine-induced prostaglandin production. The time course of prostaglandin H synthase activity in macrophages treated with staurosporine (20 nM) showed that the maximum activity was reached in 20 h. The stimulatory effect of staurosporine on thromboxane B2 production was maximum at 50 nM staurosporine and this effect was reversed at higher concentrations. Immunoprecipitation of 35S-labeled enzyme using an antibody specific for prostaglandin H synthase-2 paralleled the changes in enzyme activity. These results indicate that increased de novo synthesis of prostaglandin H synthase-2 is largely, if not solely, responsible for staurosporine-stimulated prostaglandin production in macrophages.