Cyclic 3':5' adenosine monophosphate (cAMP) mediated facilitation of neuromuscular (NM) transmission was previously implicated in the mechanisms of the reversal of nondepolarizing NM relaxants by azathioprine (AZA). This interaction of d-tubocurarine (dTC) with AZA was re-examined in rats and correlated to changes in cAMP in the same muscle. Three groups of animals were studied: controls, low-dose AZA (5 mg/kg), and high-dose AZA (50 mg/kg). After AZA or saline administration, dose-response (DR) curves for inhibition of gastrocnemius twitch tension by dTC were constructed. Contralateral gastrocnemius muscle was sampled for cAMP levels. In another group of animals, the response to 5- and 50-mg/kg boluses of AZA was recorded during a steady-state twitch depression maintained with an infusion of dTC. No significant shift in the DR curve of dTC was observed following low- and high-dose AZA. During steady-state twitch depression, high-dose AZA, however, caused a transient reversal of twitch lasting 5-10 min. High-dose AZA caused a significant (P less than 0.006) elevation of cAMP levels (340 +/- 49 pmol/mg prot) compared to control (120 +/- 18) and low-dose (163 +/- 24) AZA groups. These studies, therefore, document transient reversal of twitch tension by 50 mg/kg doses of AZA during a steady-state dTC infusion. On the other hand, AZA administered prior to dTC in low (5 mg/kg) and high (50 mg/kg) doses failed to cause a significant shift in the dTC DR curve. A three-fold increase in skeletal muscle cAMP induced by high-dose AZA does not alter dTC DR curves.