1. 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) (0.1 microM). 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 (islet-activating protein, IAP), which is known to abolish the function of GTP-binding proteins (Gi, Go and Gt), similar changes in Ca2+ transients and tension produced by treatment with Iso (0.1 microM) were noted as in non-IAP-treated preparations. However, no effects of acetylcholine (ACh; 1 microM) 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 microM), and cyclic GMP derivatives (1 mM) did not change the altered relation. In the IAP-treated preparations, ACh (1 microM) did not influence the relation altered by Iso (0.1 microM). 6. Cyclic GMP derivatives (1 mM) did not alter the Iso (0.1 microM)-increased CCR measured by perturbation analysis. ACh (1 microM) 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.