Agents known to disrupt intraterminal Ca2+ buffering, N,N-dimethylamino-8-octyl-3,4,5-trimethoxybenzoate (TMB-8), 25 microM; caffeine, 7.5 mM; N,N-bis(3,4-dimethoxyphenethyl)-N-methylamine (YS035), 180 microM; ouabain, 200 microM; and dantrolene, 50 microM, were tested for the ability to alter effects of methyl mercury (MeHg) on spontaneous quantal release of acetylcholine (ACh) at the rat neuromuscular junction. In particular, we sought to determine whether any of the above agents could prevent the MeHg-induced increase of spontaneous release of ACh, an effect measured electrophysiologically as increased frequency of miniature end-plate potentials (MEPPs). MEPPs were recorded continuously from myofibers of the rat hemidiaphragm using conventional, intracellular recording techniques during pretreatment with an inhibitor of Ca2+ regulation and subsequently with the inhibitor plus MeHg (100 microM). When given alone, caffeine and ouabain, which release Ca2+ from the smooth endoplasmic reticulum and mitochondria, respectively, increased MEPP frequency in a biphasic manner. Following pretreatment, concomitant application of MeHg with caffeine or ouabain increased MEPP frequency after a brief latent period to peak values of 53 and 92 Hz, respectively. TMB-8 and dantrolene, putative inhibitors of Ca2+ release from smooth endoplasmic reticulum, differed in their effects on MEPP frequency; TMB-8 alone decreased MEPP frequency to approximately 10% of drug-free control, whereas dantrolene did not significantly alter control MEPP frequency. Subsequent concomitant application of MeHg with TMB-8 or dantrolene increased MEPP frequency to peak values of 40 and 100 Hz after 17 and 30 min, respectively. YS035, a putative inhibitor of mitochondrial uptake and release of Ca2+, decreased MEPP frequency to less than 10% of control after 15 min when given alone. Application of MeHg following YS035 pretreatment failed to increase MEPP frequency for up to 90 min. YS035 did not mask a MeHg effect by blocking postsynaptic sensitivity to ACh or preventing its release since subsequent treatment with La3+ (2 mM) after YS035 had abolished spontaneous release, increased MEPP frequency within 5 min. Thus, of the five inhibitors of nerve terminal Ca2+ regulation tested, only YS035 prevented the stimulatory action of MeHg on MEPP frequency. Results of the present study suggest that release of Ca2+ from nerve terminal mitochondria contributes to the increased MEPP frequency caused by MeHg while release of Ca2+ from smooth endoplasmic reticulum may not.