The differential action of neuromodulators on synapses of various efficacy provides additional fine tuning of synaptic regulation beyond frequency induced plasticity. We used the well-characterized high- and low-output motor nerve terminals, of the tonic and phasic neuromuscular junctions (NMJs) in the walking leg extensor muscle of the crayfish, to investigate differential actions of serotonin (5-HT) since both terminals innervate the same target. The excitatory postsynaptic potentials of the tonic NMJ are enhanced to a greater extent than for the phasic NMJs during exposure to 5-HT (100 nM). Macropatch current recordings at identified sites along the motor nerve terminals and quantal analysis indicate that mean quantal content is substantially increased by 5-HT. The overall probability of vesicular release increases to a greater extent at tonic terminals than at phasic terminals when exposed to 100 nM 5-HT. Measures in the area (i.e. charge) of spontaneous quantal currents indicate no difference in postsynaptic receptivity to the glutamatergic synaptic transmission upon exposure to 5-HT. The results provide new details concerning differential modulation of low- and high-output synapses present on the same target tissue.