1. The effects of the immunosuppressants FK506 and rapamycin were examined in mechanically skinned skeletal muscle fibres of rat in order to determine whether the FK506-binding protein plays a role in the coupling between the voltage sensors and the Ca2+ release channels. 2. Both FK506 (1 microM) and rapamycin (1 microM) rapidly and reversibly potentiated Ca2+ release evoked by either depolarization of the transverse tubular system or caffeine application, suggesting a direct effect of the agents on the Ca2+ release channels. 3. In addition, repeated depolarizations in the presence of either FK506 (1 microM) or rapamycin (1 microM) caused irreversible loss of depolarization-induced Ca2+ release, without preventing direct activation of the Ca2+ release channels by caffeine or low [Mg2+]. If a fibre was exposed to either immunosuppressant for a similar period (10 min) without stimulation, or if the voltage sensors were kept inactivated, there was little if any loss of coupling. 4. The loss of coupling was faster at higher drug concentrations, with 20 microM rapamycin causing 50% inhibition in 7-8 min without stimulation; this was further accelerated by repeated depolarizations in the presence of the drug, but was not noticeably altered by direct activation of the release channels by repeated exposure to caffeine. The irreversible loss of coupling indicates that the FK506-binding protein may play a vital role in enabling the voltage sensors to activate the Ca2+ release channels.