Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K(+), suggesting that it is regulated not only by K(+) conductances but by inward conductances such as Na(+) and/or Ca(2+). We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na(+) with equimolar tetraethylammonium or Ca(2+) with Mn(2+) inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na(+) with N-methyl-D-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (bI(NSCC)) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of bI(NSCC). In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle.