To elucidate the mechanisms responsible for the increase in α1-adrenergic receptors during ischemia in vivo, we developed a procedure for measuring α1-adrenergic receptors in isolated, calcium-tolerant adult canine myocytes. Specific [3H]prazosin binding was rapid, saturable, reversible, and demonstrated the expected order of potency and stereospecificity for the α1-adrenergic receptor. Myocytes exposed to 30 minutes of hypoxia at 25±C or only 10 minutes at 37±C exhibited a twofold to threefold increase in the number of α1-adrenergic receptors with no significant change in receptor affinity. This hypoxia-induced increase in receptor number was reversible by 10 minutes of reoxygenation at 37±C. In contrast, more prolonged hypoxia of 80 minutes or hypotonic shock actually decreased receptor number below normoxic, control values. The concentration of long-chain acylcarnitines in myocytes also increased threefold on exposure to 30 minutes of hypoxia. Sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA, 10 μM), a potent inhibitor of carnitine acyltransferase I, not only abolished the accumulation of long-chain acylcarnitines but also the increase in α1-adrenergic receptor number induced by 30 minutes of hypoxia. Likewise, incubation of normoxic cells with exogenous palmitoyl carnitine (1 μM) for 10 minutes also increased α1-adrenergic receptor number in the presence or absence of POCA. Thus, hypoxia results in an increase in α1-adrenergic receptors associated with an increase in endogenous long-chain acylcarnitines. Furthermore, inhibition of carnitine acyltransferase I prevents not only the sarcolemmal accumulation of long-chain acylcarnitines but also the exposure of the α1-adrenergic receptor, indicating that accumulation of endogenous long-chain acylcarnitines is critical to the hypoxia-induced increase in α1-adrenergic receptors on adult myocytes. (Circulation Research 1987;61:735-746)