We examined the mechanisms underlying tachyphylaxis to beta-adrenergic receptor agonists (beta-agonists) in tracheal smooth muscle. Simultaneous measurements of isometric tension and intracellular Ca2+ concentration ([Ca2+]i) using fura-2-loaded guinea pig tracheas showed that the inhibitory effects of isoproterenol (ISO) on tension and increases in [Ca2+]i induced by methacholine exhibited marked tachyphylaxis with repeated exposure to ISO at intervals of 15 min. Similarly, the activation of single Ca2+-activated K+ (KCa) channels in on-cell patches by 1 microM ISO was gradually attenuated after repeated extracellular application of ISO to single smooth cells of porcine tracheas. Desensitization of beta-adrenergic receptor/KCa channel stimulatory coupling and relaxation responses was prevented by separately antagonizing the voltage-dependent Ca2+ channel (VDCC) with verapamil, suggesting a surprising relationship between Ca2+ influx through VDCC and beta-adrenergic desensitization. Conversely, repeated exposure of 10 U/ml protein kinase A to inside-out patches did not result in desensitization of channel activation, and repeated exposure to 10 microM forskolin modestly augmented the inhibitory effects of forskolin on tension and [Ca2+]i by methacholine, indicating that the mechanism of desensitization is mediated by the beta-adrenergic receptor/G protein complex. These results indicate that an uncoupling of beta-adrenergic receptor from KCa channels augments Ca2+ mobilization through VDCC and stimulates tachyphylaxis.