Experiments have been performed using guinea-pig and bovine trachealis in order to determine whether cromakalim and RP 49356 share the same relaxant action and to analyse the mechanisms underlying this action. RP 49356 was approximately 3 times less potent than cromakalim in suppressing the spontaneous tone of guinea-pig trachea and, like cromakalim, was antagonised by glibenclamide and by phentolamine. Biochemical studies showed that relaxant concentrations of cromakalim and RP 49356 did not alter the cAMP or cGMP content of guinea-pig trachealis muscle and did not inhibit cAMP or cGMP hydrolysis by tracheal homogenates. Like cromakalim, RP 49356 caused marked hyperpolarisation of guinea-pig trachealis cells. Patch clamp recording using inside-out membrane patches from bovine trachealis showed that cromakalim, RP 49356, glibenclamide and phentolamine were each without effect on the open state probability (Popen) of large conductance, Ca(2+)-activated K(+)-channels. We conclude that cromakalim and RP 49356 share a similar action in opening K(+)-channels in the trachealis cell membrane. This action probably does not involve the intracellular accumulation of cyclic nucleotides and the channel involved is not the large conductance, Ca(2+)-dependent K(+)-channel.