We examined the mechanisms underlying tachyphylaxis to -adrenergic receptor agonists (-agonists) in tracheal smooth muscle. Simultaneous measurements of isometric tension and intracellular Ca²⁺ ([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⁺ (K_Ca) channels in on-cell patches by 1 µM ISO was gradually attenuated after repeated extracellular application of ISO to single smooth cells of porcine tracheas. Desensitization of -adrenergic receptor/K_Ca channel stimulatory coupling and relaxation responses was prevented by separately antagonizing the VDCC with verapamil, suggesting a surprising relationship between Ca²⁺ influx through volatage-dependent Ca²⁺ channels (VDCC) and -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 µM forskolin modestly augmented the inhibitory effects of forskolin on tension and [Ca²⁺]_i by methacholine, indicating that the mechanism of desensitization is mediated by upstream of adenylyl cyclase activation. These results indicate that an uncoupling of -adrenergic receptor from K_Ca channels augments Ca²⁺ mobilization through voltage-dependent Ca²⁺ channels and stimulates tachyphylaxis.