The effect of protein kinase C activators and inhibitors on histamine-stimulated phospholipase C in bovine adrenal medullary cells has been investigated. The protein kinase C activators, phorbol 12,13-dibutyrate (PDB) or sn-1,2-dioctanoylglycerol (DOG), inhibited histamine-stimulation of phospholipase C. This inhibition was prevented by the protein kinase C-selective inhibitor Ro 31-8220 (3-[1-[3-(2-isothioureido) propyl]indol-3-yl]-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dio ne) but not the broad spectrum protein kinase inhibitor staurosporine. Indeed staurosporine on its own inhibited both the histamine-stimulated response and, in permeabilized cells, phospholipase C activated by Ca2+. Staurosporine inhibition of phospholipase C is unlikely to be mediated via protein kinase A or Ca2+/calmodulin-dependent protein kinase because it was not reproduced by selective inhibition of these kinases. Staurosporine treatment, however, reduced inositol phospholipid levels in stimulated cells. Thus staurosporine and Ro 31-8220, two widely used protein kinase C inhibitors, have quite different effects on phospholipase C activation. Furthermore, staurosporine may cause this inhibition through a reduction in the level of phospholipase C substrate.