1. In single, enzymatically dissociated, rat pancreatic acinar cells both ACh stimulation and IP3 (inositol 1,4,5-trisphosphate) injection can evoke Ca(+)-dependent transient current responses. However, exogenously applied IP3 (10 microM) gradually loses its ability to induce the Ca2(+)-dependent response (an increase in [Ca2+]i) during cell incubation with a saline solution. 2. Administration of IP4 (inositol 1,3,4,5-tetrakisphosphate, 10 microM) together with the IP3 (the injection of IP3-IP4 mixture) allows partial recovery of the response, but not full replication of the response induced by ACh (0.2 microM). Injection of IP4 alone never induces the current response. 3. The sensitivity of IP3 recovers after short-term administration of ACh (0.2 microM), and in turn, the ACh-induced response is augmented by the presence of internal IP3. These results suggest that a synergism between IP3 and another ACh-induced substance plays an important role in muscarinic Ca2+ signalling. 4. ACh-induced responses are inhibited by pre-incubation (10 min) with an activator of protein kinase C, TPA (12-O-tetradecanoylphorbol-13-acetate, 16 nM), or augmented by pre-incubation (10 min) with an inhibitor, H-7 (1-(5-isoquinoline-sulphonyl)-2-methylpiperazine, 10 microM), whilst IP3-induced responses are unaffected by that with both agents. These results indicate that protein kinase C acts negatively on the signalling elements prior to the formation of IP3. 5. The oscillatory responses, induced by cell dialysis with a nominally Ca2(+)-free (ca 1-10 microM) solution containing GTP gamma S (100 microM), are unaffected by the pre-treatment with TPA or H-7. In addition, these responses and/or those triggered by short-term stimulation with ACh and internal GTP gamma S are not influenced by external ACh. On the other hand, the oscillatory responses recorded in acinar cells pre-treated with H-7 are tightly controlled by external ACh. 6. Taken together these results suggest that activation of protein kinase C does not affect the activity of GTP-binding protein, but disconnects the link between the muscarinic ACh receptor and GTP-binding protein, or inhibits ACh binding to the receptor, in rat pancreatic acinar cells.