We have studied the mechanisms of paired-pulse facilitation (PPF) of neurotransmitter release in isolated nerve-muscle preparations of the frog cutaneous pectoris muscle. In normal extracellular Ca(2+) concentration ([Ca(2+)](o), 1.8 mM), as the interpulse interval was increased from 5 to 500 ms, PPF decayed as a sum of two exponential components: a larger but shorter first component (F1) and a smaller but more prolonged second component (F2). In low [Ca(2+)](o) (0.5 mM), both F1 and F2 increased, and a third "early" component (Fe) appeared whose amplitude was larger and whose duration was shorter than F1 or F2. In the presence of the "fast" Ca(2+) buffer BAPTA-AM, Fe disappeared, whereas F1 and F2 decreased in amplitude and duration. In contrast, the "slow" Ca(2+) buffer EGTA-AM caused a decrease of Fe and reduction or complete blockade of F2, without any changes of F1. In solutions containing Sr(2+) (1 mM), the magnitude of Fe was decreased, F1 was significantly reduced and shortened, but F2 was unaffected. Application of the calmodulin inhibitor W-7 (10 microM) at normal [Ca(2+)](o) produced a marked decrease of F2, and at low [Ca(2+)](o), a complete blockade of Fe. These results suggest that PPF at frog motor nerve terminals is mediated by several specific for different PPF components intraterminal Ca(2+) binding sites, which trigger neurotransmitter release. These sites have a higher affinity for Ca(2+) ions and are located farther from the release-controlling Ca(2+) channels than the Ca (2+) sensor that mediates phasic release.