We have used the expression of human acetylcholine muscarinic receptor (mAChR) genes in NIH 3T3 cells as a model for dissecting the molecular basis of cellular transformation induced by G protein-coupled receptors. Those mAChR subtypes efficiently coupled to PIP2 hydrolysis (m1, m3 and m5) induced agonist-dependent cell transformation whereas those inhibiting adenylyl cyclase (m2, m4) lack transforming activity. In the present study, we demonstrate that in cells expressing m1 but not m2 mAChRs the cholinergic agonist (carbachol) is alone as potent a stimulant for DNA synthesis as platelet-derived growth factor (PDGF) or serum. Furthermore, induction of DNA synthesis is shown to correlate with activation of PIP2 hydrolysis but not with inhibition of adenylyl cyclase. We also examined the role of protein kinase C (PKC) in mitogenic signalling through m1 mAChRs, and found that NIH 3T3 cells express PKC-alpha and PCK-zeta as the only conventional or Ca(2+)-independent PKC isozyme, respectively. Prolonged treatment with TPA depleted cells of PKC-alpha but not of PKC-zeta. In TPA-treated NIH 3T3 cells, the mitogenic response to a subsequent stimulation with TPA was absolutely abolished, but the response to PDGF or serum was not. Moreover, PKC depletion did not decrease DNA synthesis induced by carbachol. We conclude that carbachol potently induces reinitiation of DNA synthesis through the activation of transforming mAChR subtypes, independently of inhibition of adenylyl cyclase and conventional PKCs.