1. The heart of the nudibranch mollusc Archidoris montereyensis is uniquely responsive to regulation by identifiable cardiac motor neurons. The neurotransmitters mediating the strong excitatory and inhibitory actions of the neurons are unknown. 2. In this study we developed an infused, in vitro preparation of the Archidoris heart to determine which of several cardioactive transmitters described in mollusks could affect changes in the rate, amplitude, or tonus of cardiac contractions. Several neurotransmitters we tested increased the rate and amplitude of heart contractions, including serotonin (threshold < 10 nM), dopamine (100 nM), and the neuropeptides R15 alpha 2 (3 nM), small cardioactive peptide B (10 nM), and FMRFamide (20 microM). Myomodulin also excited the heart (0.8 microM) and potentiated the cardioexcitatory action of serotonin at subthreshold concentrations. 3. Only acetylcholine (10 nM) inhibited the heart, decreasing the rate, amplitude, and tonus of contraction. Glycine and the peptides substance P and R15 alpha 1 had no effect on the heart. 4. Antisera against the active neurotransmitters labeled central neurons and nerves innervating the heart in a pattern consistent with their putative cardioregulatory functions. 5. Thus, despite the simplicity of the cardiac motor circuit in Archidoris, contractile activity of the heart appears to be regulated by several neurotransmitters, each with subtly different modes and thresholds of action.