Sustained contractions of smooth muscle cells (SMC) maintain basal tone in the internal anal sphincter (IAS). To examine the molecular bases for the myogenic tone in the IAS, present studies focused on the role of RhoA/ROCK in the SMC isolated from the IAS versus the adjoining phasic tissues of the rectal smooth muscle (RSM) and anococcygeus smooth muscle (ASM) of rat. We also compared cellular distribution of RhoA/ROCK, levels of RhoA-GTP, RhoA-Rho GDI complex formation, levels of p^Thr696-MYPT1 and SMC relaxation caused by RhoA inhibition. Levels of RhoA/ROCK were higher at the cell membrane in the IAS SMC as compared with those from the RSM and ASM. C3-exoenzyme (RhoA inhibitor) and Y 27632 (ROCK inhibitor) caused a concentration-dependent relaxation of the IAS SMC. In addition, active ROCK-II (primary isoform of ROCK in SMC) caused further shortening in the IAS SMC. C3-exoenzyme increased RhoA-Rho GDI binding and reduced the levels of RhoA-GTP and p^Thr696-MYPT1. ROCK inhibitor attenuated PKC-induced contractions in IAS SMC. Conversely, PKC inhibitor (Gö 6850) (that causes partial relaxation of the SMC) had no significant effect on ROCK-II induced contractions. Further experiments showed highest levels of RhoA, active form of RhoA (RhoA-GTP), ROCK-II, MLC₂₀, and of phospho- MYPT1, and -MLC₂₀ in the IAS SMC vs. RSM and ASM. However, the trend was reverse with the levels of inactive RhoA (GDP-RhoA-Rho GDI complex), and MYPT1. We conclude that RhoA/ROCK play critical role in maintenance of spontaneous tone in the IAS SMC via inhibition of myosin light-chain phosphatase.