The effects of streptomycin on neuromuscular transmission were investigated on frog cutaneous pectoris muscles. The half-inhibition doses of peak end-plate current amplitude are 3 x 10(-5) and 8.5 x 10(-5) M in the presence of 0.9 and 1.8 mM extracellular calcium, respectively. The quantal content of the end-plate current was reduced by 50% in the presence of 3 x 10(-5) M streptomycin in Ringer's solution containing 0.35 mM Ca and 2 mM Mg. Miniature end-plate currents under these conditions were reduced by only 20%, suggesting that the presynaptic blocking action predominates over the postsynaptic action. A much higher concentration of streptomycin (3.5 x 10(-4) M) was required to achieve 50% block of peak transient depolarizations induced by iontophoretic application of acetylcholine. The postsynaptic action involves primarily a blocking action on acetylcholine receptors since the drug did not alter the linearity of current-voltage relationship for end-plate currents at membrane potentials more positive than -50 mV. An additional weak blocking action on the acetylcholine-activated channels exhibited a slight voltage and concentration dependence, giving rise to a slight prolongation of the end-plate current and curvature of the current-voltage relation at membrane potentials more negative than -50 mV. Thus, under normal conditions the predominant action of streptomycin at the neuromuscular junction is to reduce transmitter release. A secondary competitive inhibition on the acetylcholine receptor and a weak blocking action on the ionic channels may also contribute to the overall block.