Identifiable neurons in the marine mollusc, Aplysia californica, were used to improve isolation techniques for in tandem physiological and chemical studies of the same neuron. A previous study demonstrated that the histaminergic neurons, RC-2 and LC-2, which had been subjected to intracellular recording and staining, were particularly sensitive to a freeze-substitution procedure used in isolating neurons for subsequent chemical assay. The present report describes an improvement of the freeze-substitution procedure which permits excellent recovery of histamine in physiologically manipulated C-2 neurons. The in tandem physiological and chemical procedures developed for identified neurons were used to characterize additional histamine-containing neurons which cannot be identified by visual inspection alone. These new histaminergic neurons, designated RC-3 and LC-3, are located on the ventral surface of the cerebral ganglion and are distinguishable from other adjacent histamine-containing neurons by their monosynaptic connections with some of the same follower neurons previously described for RC-2 and LC-2. The results of this study further support the role of histamine as a multiaction neurotransmitter and demonstrate the feasibility of measuring endogenous levels of various neurotransmitters in neuronal somata subsequent to intracellular recording.