We are investigating peptidergic neurotransmission between ganglia in the Aplysia CNS. Neurons in the cerebral ganglia of Aplysia synthesize [35S]methionine-labeled peptides, which are transported to other central ganglia by fast axonal transport. We report the characterization of one of these peptides, termed cerebral peptide 1 (CP1). Transported [35S]methionine-labeled CP1 was used as a probe for the purification of CP1 from the pooled extracts of 1000 cerebral ganglia using three sequential modes of RP-HPLC. Amino acid sequence analysis by automated Edman degradation yielded the following sequence: Phe-Ser-Gly-Leu-Met-Ser-Glu-Gly-Ser-Ser-Leu-Glu-Ala. This sequence was consistent with data from amino acid composition analysis and FAB-MS of the purified peptide. In addition, a synthetic peptide with the proposed sequence coeluted with [35S]methionine-labeled native CP1 using the three RP-HPLC conditions used in the purification. Antisera raised against synthetic CP1 stained about 90 neuronal cell bodies in the cerebral ganglion including those of the asymmetric H cluster. Extracts of the H cluster dissected from ganglia incubated in [35S]methionine synthesized a radiolabeled peptide that coeluted with synthetic CP1, indicating that these neurons indeed synthesize authentic CP1. The other major ganglia each contained only a few CP1-immunoreactive neurons. The neuropil of each ganglion contains both smooth and varicose-immunoreactive fibers. Thus, CP1 is broadly distributed within the Aplysia CNS and may be a new peptide transmitter.