The presence of peptides in pure cultures of neurons from 8-day-old chick embryo cerebral hemispheres has been investigated by means of specific radioimmunoassays and chromatographic purification. Somatostatin, Met-enkephalin, Leu-enkephalin, and substance P immunoreactive substances have been detected in 8-day-old cultures grown in serum-free culture medium. The peptides were present in the cellular extracts, as well as in the culture medium extracts. beta-Endorphin, thyroliberin, luteinizing hormone-releasing hormone, and ACTH could not be detected. The largest amount was accounted by somatostatin (48 +/- 2 ng/mg protein). Some 60% of the somatostatin-immunoreactive material was found in the culture medium. Met-enkephalin, Leu-enkephalin, and substance P were present at lower concentrations: 1.61 +/- 0.27, 0.24 +/- 0.02, and 0.14 +/- 0.005 ng/mg protein, respectively. The identities of somatostatin- and enkephalin-immunoreactive materials were confirmed by high pressure liquid chromatography. The findings suggest that cultured neurons that express dopaminergic and GABAergic properties contain peptides similar, if not identical, to somatostatin, Met-enkephalin, Leu-enkephalin, and substance P.