SUMMARY1. We investigated the mechanism of signal transduction during the effect of muscarinic receptor stimulation on Ca2+ transients, tension, Ca2+ sensitivity and the cross-bridge cycling rate (CCR).2. Membrane-permeable derivatives of cyclic GMP (8-bromo-cyclic GMP and dibutyryl cyclic GMP) did not cause any significant changes in the peaks of Ca2+ transients and tension and the time courses of either signal modulated by isoprenaline (Iso) (01 /tM).3. Nitroprusside (0 1-1 mM) likewise did not change the peaks or the time courses of Ca2+ transients and tension in the Iso-treated preparations.4. In papillary muscles excised from ferrets treated with pertussis toxin (isletactivating protein, IAP), which is known to abolish the function of GTP-binding proteins (Gi, G. and Gt), similar changes in Ca2+ transients and tension produced by treatment with Iso (0-1 /M) were noted as in non-IAP-treated preparations. However, no effects of acetylcholine (ACh; 1 aUM) on either signal were observed.5. The relation between [Ca2+]i and tension measured during the steady state of tetanic contraction was shifted to the right by Iso (0-1 /tM), and cyclic GMP derivatives (1 mM) did not change the altered relation. In the IAP-treated preparations, ACh (1 /tM) did not influence the relation altered by Iso (01 /IM).6. Cyclic GMP derivatives (1 mM) did not alter the Iso (01 /tM)-increased CCR measured by perturbation analysis. ACh (1 /tM) did not restore the Iso-increased CCR in the IAP-treated preparations.7. These results suggest that signal transduction in muscarinic receptor stimulation is primarily mediated by inhibition of adenylate cyclase via IAP-sensitive GTP-binding proteins, and that cyclic GMP does not play an important role in the effect of muscarinic receptor stimulation on Ca2+ transients, tension, Ca2+ sensitivity or CCR.