The effects of d-tubocurarine (dTC) on nicotinic synapses on sympathetic B and C neurons in the bullfrog were compared by recording trains of postganglionic compound action potentials (CAPs) at 0.5–20 Hz. Block by dTC was strongly use dependent in that the IC50 shifted almost 10-fold with increasing stimulus frequency. Maximum sensitivity to dTC occurred at 5 Hz in the B system and at 20 Hz in the C system. Recovery during posttrain periods was characterized by transient reduction of CAP amplitude in the B system and by transient enhancement of CAP amplitude in the C system. Thus, dTC distinguished between nicotinic synapses on the two cell types. The cell-specific effects of dTC could arise from differences in postsynaptic or presynaptic nicotinic receptors, or from differences in acetylcholine (ACh) release. We tested these possibilities using intracellular recording. Based on comparison with iontophoretic responses to ACh, changes in EPSP amplitude during repetitive stimulation in dTC could not be explained by altered postsynaptic sensitivity. The block of nicotinic receptors was further analyzed by recording synaptic currents. In B and C cells, 3 microM dTC competitively antagonized EPSC amplitude by 54% without any sign of open-channel block. In B cells, comparison of trains in normal Ringer and dTC revealed a constant fractional reduction in EPSC amplitude, thereby indicating that presynaptic nicotinic receptors do not influence release. In the C system, dTC had no effect on peptidergic EPSPs, thereby suggesting that presynaptic nicotinic receptors also do not influence peptide release. We conclude that the differential effects of dTC on nicotinic transmission are indirect consequences of differences in ACh release by preganglionic B and C neurons.