BACKGROUND: The potent bronchodilatory effects of ketamine on airway smooth muscle tone are important in the management of patients with asthma, but its mode of action is unclear. In the present study we evaluated that effects on isolated guinea pig tracheal smooth muscle. METHODS: Changes of isometric contraction of strip were measured. (1) Serial stimulation with acetylcholine(ACh) in Krebs solution or with A23187, nifedipine, ketamine were evaluated. After that, ACh stimulation was induced in Ca2+ free solution. (2) In Ca2+ free solution, ACh contraction was obtained(L1) and emptied by repetitive ACh stimulation. Internal stores were refilled by Ca2+ with ACh stimulation. During the incubation period, A23187, nifedipine, ketamine, cyclopiazonic acid + ketamine was added and tested for their ability to inhibit refilling. Refilling was evaluated by ACh produced contraction (L2) with ratio (L2/L1). (3) Effects of ketamine on the contraction induced by caffeine were also checked. RESULTS: Ketamine inhibited amplitude dose-dependently by successive application of ACh in modified Krebs solution and Ca2+ free solution. Ca2+ influx through voltage gated channels were inhibited with nifedipine but not with A23187. ACh sensitive internal store were different when A23187, nifedipine and ketamine were applied in Ca2+ free solution. Refilling of internal store were potentiated by A23187, but decreased by nifedipine and ketamine. Caffeine produced contractions in the presence of ketamine were not significantly different from control. CONCLUSION: We concluded that the inhibitory effects of ketamine in guinea pig trachea were by acting through voltage and receptor gated channels in dose-depedent manner and these effects may be interferences of intracellular second messengers system.