The effect of 2,4-dinitrophenol (DNP) was investigated at the frog neuromuscular junction to elucidate the mechanism by which mitochondrial inhibitors increase transmitter release. After a prolonged lag time (30 min), DNP (10(-4) M) caused an increase in miniature endplate potential frequency (MEPP F), which peaked within 10 min and then declined toward, but not reaching, control values. The time course and peak effect of the response were not altered by Na and Ca channel blockade [produced by 3 microM tetrodotoxin (TTX) plus 1.8 mM Co++], but the lag time was sufficient to prevent influex of ions at the nerve terminal, as the same concentration caused complete block of a 50-fold MEPP F increase to 15 mM [K+]. The results suggest that mitochondrial inhibitors produce their effect primarily by reversing the Ca++ uniporter and not by a Na+-mediated stimulation of the Na+-Ca++ antiporter. Their mechanism in this regard differs from that underlying the MEPP F increase produced by cardiac glycosides. The long lag time with DNP may be due to Na pump inhibition and nerve terminal swelling, and the shortening of lag time by TTX plus Co++ to an inhibition of Na+ influx and swelling. Finally, the block of MEPP F with TTX plus Co++ indicates that the MEPP F increase with Co++ alone is probably due to entry of Co++ through unblocked Na channels.