In order to explore the functions of the peptide proctolin in the lobster nervous system, 3 classes of neurons showing proctolin-like immunocytochemical staining were selected for study. These neurons were identified on the basis of physiological and/or morphological criteria, isolated by dissections, and analyzed with biochemical methods to determine whether they contained authentic proctolin and which classical neurotransmitters coexisted with the peptide. Pairs of large proctolin-immunoreactive neurons in fifth thoracic and first abdominal ganglia were identified as serotonin-immunoreactive neurons (Beltz and Kravitz, 1983, 1987) by staining serial sections of the ganglia alternately with the 2 antisera. Physiologically identified cells, dissected from the ganglia and analyzed with high-performance liquid chromatography (HPLC), contained approximately 20 microM proctolin and 0.5 mM serotonin. A large proctolin-immunoreactive neuron in the circumesophageal ganglion was identified as the lobster homolog of a dopaminergic neurosecretory cell found in other crustaceans (Cooke and Goldstone, 1970). The large lobster cell stained with antityrosine hydroxylase antiserum, and synthesized 3H-dopamine from 3H-tyrosine. Dissected cell bodies, analyzed by HPLC, contained approximately 25 microM proctolin. Proctolin-immunoreactive sensory neurons were identified as large stained fibers that terminated in sensory dendrites of the oval organ mechanoreceptor in the scaphognathite (Pasztor, 1979; Pasztor and Bush, 1982). The largest sensory fiber was isolated for biochemical studies. It synthesized 3H-acetylcholine from 3H-choline and, by HPLC analysis, was found to contain approximately 3 microM proctolin. Thus, proctolin coexists with different conventional transmitters in several classes of identified lobster neurons. Investigations of the actions of proctolin in these different contexts should contribute to a more complete understanding of the diverse functions of neuropeptides and their roles as cotransmitters.