The ventral nerve cord of crayfish contains axons of five pairs of excitatory interneurons, each of which can activate the swimmeret system. Perfusion of the ventral nerve cord with the neuropeptide proctolin also activates the swimmeret system. The experiments reported here were conducted to test the hypothesis that one or more of these excitatory interneurons uses proctolin as a transmitter. Each of the five excitatory axons was located and stimulated separately in an individual crayfish, and similar motor activity was elicited by stimulating each of them. Quantitative comparison of spontaneous swimmeret motor patterns with activity caused by stimulating one of these excitatory axons, EC, or by perfusing with proctolin solutions showed that the motor patterns produced under these three conditions were not significantly different (P > 0.05). By using a new, affinity-purified proctolin antiserum, we labeled axons in the connective tissue between the last thoracic and first abdominal ganglion and compared the positions of labeled axons with the previously described positions of the excitatory axons. About 0.3% of the axons in these connective tissues showed proctolin-like immunoreactivity, but heavily labeled pairs of axons did occur bilaterally in the regions of excitatory swimmeret axons. The projections of these labeled axons into the abdominal ganglia were traced in serial plastic sections. Labeled processes were abundant in the lateral neuropils, the loci of the swimmeret pattern-generating circuitry. From this evidence, we propose that three of these excitatory swimmeret interneurons use proctolin as a transmitter, but that a fourth does not. The evidence for the fifth axon is ambiguous.