Visceral smooth muscles co-express two muscarinic receptor subtypes, M2 and M3, which produce two major effects during cholinergic excitation – adenylyl cyclase inhibition and PLCβ activation, respectively. In our work we studied the relevance of these effects for muscarinic cationic current (mI_CAT) generation, which underlies cholinergic excitation. Experiments were performed on single ileal cells, from humanely killed guinea-pigs, using patch-clamp techniques with weakly buffered [Ca²⁺]_i (50 μM EGTA) or with [Ca²⁺]_i ‘clamped’ at 100 nM (10 mM BAPTA/CaCl₂ mixture). Application of cAMP-elevating agents (1 μM isoproterenol or 10 μM 8-Br-cAMP) had no effect on mI_CAT. In contrast, we found that PLCβ plays one of the major roles in the regulation of mI_CAT. Firstly, InsP₃-induced Ca²⁺ release due to M3 receptor activation facilitates mI_CAT, an effect which could be inhibited by the PLC blocker U-73122. Secondly, PLCβ activation was important for cationic current generation even when [Ca²⁺]_i was ‘clamped’ at 100 nM. U-73122 inhibited mI_CAT with an IC₅₀ of 0.72 ± 0.18 μM (n=4) by reducing the maximal conductance and shifting the activation curve positively and at 2.5 μM virtually abolished both carbachol- and GTPγS-induced cationic current (n=18). InsP₃ (1 μM, n=3) in the pipette or OAG (20 μM, n=7) applied externally had no effect on mI_CAT or its inhibition by U-73122. U-73343, a structural U-73122 analogue which inhibits PLC only weakly, had no effect on GTPγS-induced mI_CAT (n=4), but weakly inhibited carbachol-induced current (n=5), possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mM (n=3). Aristolochic acid (n=3), which inhibits PLA2, as well as D-609 (n=7) and NCDC (n=4), which inhibit phosphatidylcholine-specific PLC had no or very small effect on mI_CAT, suggesting that these enzymes were not involved. It is concluded that the ‘permissive’ role of the M3 receptor subtype in mI_CAT generation is exerted via PLCβ-dependent modulation of mI_CAT both through Ca²⁺ release and independently of InsP₃, DAG, Ca²⁺ store depletion or a rise of [Ca²⁺]_i.