The distribution of substance P-like immunoreactivity (substance P-li) in the central nervous system of Limulus polyphemus was studied by using indirect immunocytochemical techniques. Six bilaterally symmetrical pairs of cell clusters in the circumesophageal connectives and the subesophageal mass contain substance P-li. Two of those pairs are the source of a system of efferent fibers that is involved in the expression of circadian rhythms of photosensitivity by the lateral eye. Substance P-li-containing cells were also observed scattered along the length of the circumesophageal connectives, which contain abundant stained fibers and some terminals. Substance P-li fibers leave through the ventral and dorsal nerves of the posterior circumesophageal ring. The neuropil of the subesophageal mass contains an abundance of stained terminals. Immunoreactive fibers can be seen throughout the length of the two longitudinal connectives of the ventral cord, in discrete fiber tracts in the lateral edges of the interganglionic connectives, and in the dorsal and ventral nerves of abdominal ganglia 1-4. Each of these ganglia contains three pairs of substance P-immunoreactive cell body clusters: an anterolateral, a medial longitudinal, and a medial posterior cluster. Substance P-li fibers entering through the ventral (posterior) nerves form very distinctive fascicles in each side of the ganglia, giving off fibers throughout their length. The neuropil is filled with immunoreactive terminals distributed homogeneously. The anterolateral clusters of the abdominal ganglia may be involved in cardioregulation. The six pairs of clusters in the posterior circumesophageal ring, and perhaps some of those in the abdominal ganglia, are believed to constitute a neurosecretory system, projecting to multiple targets throughout the organism. This system is postulated to modulate various sensory inputs and motor activity, and could be driven by a circadian clock, as well as by other systems responsible for integrated organismic responses.