We compared responses of Chinese hamster ovary (CHO) cell lines stably transfected with human genes for the M1-M5 muscarinic receptor subtypes to several stimuli. While ATP brought about similar increases in the concentration of intracellular Ca2+ ions ([Ca2+]i) in the cell lines expressing all individual receptor subtypes, carbachol acted with much higher potency and efficacy on the cells expressing the M1, M3, and M5 receptor subtypes than on those expressing the M2 and M4 subtypes. The maximum [Ca2+]i responses to ATP corresponded to 41-75% of the maximum responses to carbachol in the cells expressing the M1, M3, and M5 receptor subtypes. The responses to ATP were strongly suppressed (> 75% decrease) by a preliminary administration of a maximally active concentration of carbachol in these three cell lines, whereas the responses to carbachol were less sensitive to the preliminary administration of a maximally active concentration of ATP (< 25% decrease). It appears likely that carbachol and ATP release Ca2+ ions from identical intracellular stores. Tetradecanoylphorbol acetate (TPA) strongly inhibited the responses of [Ca2+]i to both carbachol and ATP and enhanced the incorporation of [14C] choline into lipids in all five CHO cell lines investigated. On the other hand, the incorporation of [14C] choline into lipids was diminished by carbachol in the cell line expressing the M3 receptor subtype and unchanged in the other cell lines. This effect of carbachol was not dependent on the presence of extracellular Ca2+ ions and was not affected by TPA, which diminished the response of [Ca2+]i to muscarinic stimulation. It is suggested that it was due to muscarinic receptor-mediated activation of phospholipase D.