This study examined the effects of the selective Rho-kinase inhibitor Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride]) on cholinergic nerve-mediated contraction and neurotransmitter release in murine and guinea-pig isolated tracheal preparations. In tracheal preparations obtained from both species, Y-27632 shifted carbachol concentration-effect curves to the right and reduced the maximal contractile response. Repeated electrical field stimulation (EFS) evoked transient, consistent and reproducible contractions in murine and guinea-pig tracheal preparations. Y-27632 inhibited these cholinergic nerve-mediated contractions in a concentration-dependent manner. EFS (0.1-30 Hz) elicited frequency-dependent cholinergic nerve-mediated contractile responses. In murine tracheal preparations, Y-27632 (3 microM and 10 microM) shifted frequency-response curves to EFS to the right by 5.5 and 13.0 fold respectively and markedly reduced the maximal contractile response. In murine and guinea-pig tracheal preparations loaded with [(3)H]-choline, Y-27632 (10 microM) significantly increased the EFS-induced outflow of radioactivity from airway cholinergic nerves by 27% and 54% respectively. Thus, Y-27632 inhibited both carbachol-induced and cholinergic nerve-mediated contractile responses. Conversely, Y-27632 increased neurotransmitter release from airway cholinergic nerves. However, since antagonism of acetylcholine-induced contraction by Y-27632 overwhelmed the increased neurotransmitter release, the overall effect of this Rho-kinase inhibitor was to inhibit cholinergic nerve-mediated contraction.