The distribution of a substance P‐like material in turtle brain, spinal cord, dorsal root ganglion, and retina was determined using radioimmunoassay (RIA) and immunohistochemistry. High levels of a substance P‐like material were found in turtle neural tissue, particularly in basal telencephalon, hypothalamus, and tegmentum. In many regions, the concentration of a substance P‐like material in turtle nervous tissue was found to be similar, in a region‐to‐region comparison, to that previously reported for birds and mammals, particularly for the more “phylogenetically conservative” parts of the nervous system (such as the basal ganglia, tegmentum, and hypothalamus). The slopes of substance P RIA dose‐response curves for tissue extracts from nearly all regions of the turtle nervous system examined were parallel to a standard dose‐response curve for synthetic substance P (SP). The immunohistochemical results, with anti‐substance P antisera from guinea pig or rabbit, or with a monoclonal antibody, were consistent with the RIA data. Regions showing high concentration of an SP‐like material by RIA were observed to contain numerous neurons and/or fibers containing an SP‐like material. The immunohistochemical results provide evidence for the presence in turtle of numerous SP‐containing pathways, several of which (e.g., an SP‐containing strionigral pathway, an SP‐containing striopallidal pathway and an SP‐containing dorsal root ganglia‐spinal dorsal horn pathway), have been described in birds and mammals. The present results thus suggest that the neuropeptide SP has had a largely stable evolutionary history as a transmitter or modulatory agent during amniote brain evolution. Copyright © 1984 Alan R. Liss, Inc.