Immunochemical studies were carried out on the ascidian Ciona intestinalis to determine the character and distribution of the tachykinins neurokinin A (NKA) and substance P (SP). Antisera specific for the C-terminus of mammalian SP, and for the N-terminus of mammalian SP and NKA, were used to monitor tissue extracts from Ciona. Parallel immunocytochemical studies assessed the distribution of these tachykinins in the central and peripheral nervous systems as well as their occurrence in endocrine cells. HPLC and radioimmunoassay established the presence of both C-terminal SP and NKA-like material in extracts from neural ganglion and body wall/pharynx. Immunocytochemistry revealed the C-SP material to be present in a population of small neuronal cell bodies and fibers in the ganglion as well as in cell bodies and fibers in the periphery. The NKA-like material was restricted to separate and larger neuronal perikarya in the ganglion while in the periphery its distribution reflected that of the C-SP-like material. Endocrine cells in the pharyngeal epithelium were reactive only with the C-terminal SP antiserum. N-terminal SP antisera were unreactive both in radioimmunoassay and immunocytochemistry. These findings are in accord with the idea that the tachykinin family is represented by at least two of its members at the prevertebrate stage of evolution. Interestingly, the SP-like material shows strong C-terminal homology with the mammalian peptide but little N-terminal similarity. Furthermore, the NKA-like peptide is restricted to the nervous system while SP-like molecules may be found in both central and peripheral neurons as well as endocrine cells in the pharynx.