The muscarinic cholinergic receptor (mAChR) subtypes share high sequence similarity except in their third intracellular loop and COOH terminus, domains thought to be involved in signal transduction. Subtypes M1, M3, and M5 couple mainly through Galpha(q/11), and M2 and M4 couple mainly through Galpha(i/o). Whether subtypes within each of these two groups differ in their signaling pathways remains to be resolved. This study focused on nuclear signaling pathways leading to activation of the transcription factor, serum response factor (SRF). Genes encoding M1, M2, and M3 were co-expressed in Jurkat T lymphocytes with a reporter gene driven by a mutant serum response element, SRE.L, which responds to SRF activation. We show that only M1 mAChR activated SRF through a pathway involving the small GTPase RhoA, with no response observed for M2 and M3. Transfection of GTPase-deficient Galpha subunits (GalphaQL; constitutively active form) demonstrated that SRF was activated by Galpha(13)QL but only marginally by Galpha(q)QL and Galpha(12)QL in Jurkat cells. Yet transfection of regulator of G protein-signaling protein, RGS2 and RGS4, which inhibit Galpha(q/11) activity, indicated that Galpha(q/11) and Ca(2+) mobilization were required for SRF activation by M1. Calmodulin inhibitors suppressed the M1 and the Galpha(13)QL pathways, acting both upstream and downstream of RhoA. However, calcineurin inhibitors and the tyrosine kinase inhibitor genistein selectively suppressed SRF activation by M1, but not by Galpha(13)QL, indicating the presence of separate pathways. The calmodulin-dependent tyrosine kinase Pyk2 was also activated by M1 but not M3, and Pyk2 appears also to play a role in M1-SRF activation, as judged by experiments with two dominant-negative Pyk2 mutants. These results reveal a novel calmodulin-dependent RhoA-SRF signaling pathway unique to the M1 mAChR subtype.