The selectivity of different Rho kinase (ROCK) inhibitors in the spontaneously tonic smooth muscle has not been investigated. We examined this issue using Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxanecarboxamide, 2HCL], H-1152 [(S)-(+)-2-methyl-5-isoquinolinyl)sulfonyl]homopiperazine, 2HCl], HA-1077 [(5 isoquinolinesulfonyl)homopiperazine, 2HCl], and ROCK inhibitor II, [N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea]. We compared these inhibitors in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). ROCK, protein kinase C (PKC) and myosin light chain kinase (MLCK) activities were determined in the IAS, before and after different ROCK inhibitors. Y-27632 and H-1152 were ~30-fold more potent in the IAS (IC₅₀: 4.4 x 10^-7 M and 7.9 x 10^-8 M, respectively) vs. the phasic rectal smooth muscle (RSM) (IC₅₀: 1.3 x 10^-5 M and 2.5 x 10^-6 M, respectively). HA-1077 and ROCK inhibitor II were equipotent in the IAS vs. RSM. In the IAS, H-1152 was the most potent while ROCK inhibitor II being the least. Y-27632 and H-1152 caused concentration-dependent decrease in the IAS tone that correlate directly with the decreases in ROCK activity, without significant effect in the PKC and MLCK activities. This specifically selective correlation between ROCK activity and decrease in the IAS tone was absent in the case of HA-1077 and ROCK inhibitor II which also inhibited PKC and MLCK. We conclude that the IAS tone is critically dependent on ROCK activity, and H-1152 and Y-27632 are most selective and potent ROCK inhibitors in the IAS.