Purified cromakalim trans enantiomers were tested for their ability to antagonize three specific mechanisms of smooth muscle activation, i.e., depolarization-induced Ca2+ entry through voltage-gated channels, agonist-induced Ca2+ entry, and agonist-induced Ca2+ release. Cromakalim effects were studied in rabbit aortic rings contracted by stimuli corresponding to the above mechanisms. First, aortic rings were contracted by increase in extracellular [K+] (to 27 mM), which causes partial membrane depolarization. Under these conditions, (-)-cromakalim exhibited an EC50 of 0.18 microM and a 150-fold higher relaxing potency than the (+)-enantiomer. Second, in aortic rings tonically contracted by 1 microM norepinephrine (NE) in the presence of 1 microM nifedipine, i.e., in rings contracted mainly owing to NE-stimulated Ca2+ entry through receptor-operated channels, (-)-cromakalim induced relaxation with an EC50 of 0.68 microM and exhibited a 191-fold higher potency than the (+)-enantiomer. Third, phasic, NE-induced contractions of rabbit aortic rings in the absence of extracellular Ca2+, i.e., contractions that reflect release of Ca2+ from intracellular stores, were antagonized with an EC50 of 0.29 microM and a 144-fold higher potency than the (+)-enantiomer. All effects of (-)-cromakalim were blocked by either completely depolarizing the vessels with high extracellular [K+] (40 mM) or by addition of the K+ channel blocker glibenclamide (10 microM). Cromakalim relaxed rabbit aorta independent of the mechanism underlying smooth muscle tone. Cromakalim effects were equivalent with respect to dose dependence, stereoselectivity, and sensitivity to extracellular [K+] and glibenclamide.(ABSTRACT TRUNCATED AT 250 WORDS)