The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. the coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completly different effects of 5′-guanylyl imido-diphosphate [Gpp(NH)p] on muscarinic agonist binding to homogenates from young vs aged cultures [Moscona-Amir, E., Henis, Y. I., Yechiel, E., Barenholz, Y., & Sokolovsky, M. (1986) Biochemistry 25, 8118–8124]. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no such effect was observed in homogenates from young cultures. IAP-catalyzed [32P] ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-αi antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-αi or anti-α°antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. the parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. the role of the membrane lipid composition in these phenomena is discussed.