The localization of crustacean cardioactive peptide-like immunoreactivity in the horseshoe crab Limulus polyphemus was investigated with enzyme-linked immunosorbent assay and fluorescence microscopy. Immunoreactivity was quantified in the opisthosomal nervous system (67.7 +/- 11.4 ng/g), cardiac ganglion (45.0 +/- 10.3 ng/g), prosomal nervous system (28.5 +/- 6.6 ng/g), and midgut (24.6 +/- 6.7 ng/g). In the brain, immunoreactive somata were observed in ganglion cells of the central body, in the medullary group and within the ventral medial group. Clusters of immunoreactive cells were found in each of the circumesophageal, pedal ganglia, and in the opisthosomal, abdominal ganglia. In the periphery, immunoreactive varicose fibers were observed in branches of the intestinal nerves, and near longitudinal and circular muscle fibers of the midgut. Immunoreactivity was observed in the cardiac ganglion and myocardium of the neurogenic heart. Synthetic crustacean cardioactive peptide had slight excitatory effects on the cardiac rhythm at doses up to 10(-6) M. This peptide had excitatory effects on the midgut at nanomolar doses. Ventral nerve cord extracts were partially purified with reverse phase high performance liquid chromatography. Two regions of immunoreactivity were detected, one of which coeluted with the authentic peptide. The distribution of crustacean cardioactive peptide immunoreactivity is compared with other transmitter systems in the Limulus nervous system, and myotropic actions of this peptide are discussed with respect to peptidergic modulation of intestinal motility.