1. Dual‐excitation microfluorometry (Fura‐2 as indicator) was employed to monitor directly changes in the cytosolic calcium concentration [( Ca2+]i) in single cells. We investigated and compared the effects of stimulation of AR42J rat pancreatic acinar cells by two peptide agonists, substance P and bombesin. 2. Substance P (10(‐7) M) and bombesin (10(‐8) M) each gave rise to a marked, but transient, elevation in [Ca2+]i. The calcium signals evoked by the two peptides were qualitatively and quantitatively very similar. However, in the absence of extracellular Ca2+ the response to substance P, but not bombesin, was abolished. These results suggest that substance P induces calcium influx across the cell surface membrane but does not release calcium from internal stores. Bombesin in marked contrast releases calcium from intracellular stores in the absence of any detectable calcium influx. 3. Depolarization by high‐K+ extracellular solutions evoked a marked, but transient, rise in [Ca2+]i. This elevation in [Ca2+]i was strictly dependent upon the presence of Ca2+ in extracellular media. 4. Nifedipine (5 x 10(‐6) M), an antagonist of L‐type voltage‐dependent Ca2+ channels, blocked the elevations in [Ca2+]i induced by either substance P or high‐K+ solutions, but not that evoked by application of bombesin. 5. Patch‐clamp, single‐channel current recordings from cell‐attached patches of membrane confirmed the presence of voltage‐dependent calcium channels in the surface membranes of AR42J cells. Whole‐cell current recordings demonstrated voltage‐dependent inward Ca2+ (Ba2+) currents which were increased in amplitude by substance P and blocked by nifedipine. 6. The protein kinase C (PKC) activators, the phorbol diester, phorbol 1,2‐myristate 13‐acetate (PMA, 10(‐7) M), and cell‐permeable diacylglycerol analogues, 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG, 2.5 x 10(‐6) M) and sn‐2‐dioctanoyl glycerol (DiC8, 2.5 x 10(‐6) M), mimicked the effect of substance P, but not bombesin, in elevating [Ca2+]i in a manner that was blocked by removal of extracellular Ca2+ or application of nifedipine. 7. The PKC inhibitor, polymyxin B (2.5 x 10(‐6) M), applied 2 min prior to stimulation blocked the effects of substance P and PKC activators, but not bombesin, in elevating [Ca2+]i. 8. The calcium signals evoked by substance P and bombesin are achieved by activation of different molecular mechanisms. Substance P, the evidence suggests, activates PKC which in turn stimulates calcium influx by opening voltage‐dependent Ca2+ channels in the cell surface membranes.(ABSTRACT TRUNCATED AT 400 WORDS)