Earlier studies have shown that bradykinin stimulated release of catecholamines from chromaffin cells by an influx of calcium through dihydropyridine-insensitive channels, and also that bradykinin stimulated (poly)phosphoinositide hydrolysis. To investigate membrane-bound second messengers in chromaffin cells, and to elucidate any role these may play in stimulus-secretion coupling, we have studied the influence of bradykinin on diacylglycerol and phosphatidic acid (PA). Using equilibrium labelling of primary cultures of chromaffin cells with [H-3]arachidonic acid or [H-3]glycerol, we found no influence of bradykinin (10 nM) on labelled diacylglycerol formation, either in the presence or absence of inhibitors of diacylglycerol lipase or kinase. However, when we used cells prelabelled with P-32i for 2.5 h, we found that bradykinin produced a substantial stimulation of label found in PA, with an EC50 value of about 1 nM. This bradykinin stimulation of [P-32]PA formation was only partially dependent on extracellular calcium, in contrast to the smaller response to nicotine, which was completely dependent on extracellular calcium. Short (10 min) pretreatment with tetradecanoylphorbol acetate (TPA) almost completely eliminated the bradykinin-stimulated formation of inositol phosphates, but failed to affect bradykinin stimulation of label in PA, suggesting that PA production in response to bradykinin is not downstream of phospholipase C activation. TPA alone failed to stimulate [P-32]PA substantially, whereas long-term (24 or 48 h) treatment with TPA failed to attenuate the response to bradykinin. Diacylglycerol kinase inhibitors were also without effect on the bradykinin stimulation of [P-32]PA. These results suggest that bradykinin stimulates PA production by a mechanism independent of the activation of protein kinase C. A preliminary indication that G proteins may be involved was suggested by the observation that AlF4- stimulates [P-32]PA accumulation whereas N-ethylmaleimide inhibits bradykinin-stimulated [P-32]PA accumulation.