In the first 3 papers of this series (Furshpan et al., 1986a, b; Potter et al., 1986), a sensitive microculture procedure was used to show that sympathetic principal neurons, dissociated from newborn or adult superior cervical ganglia and grown singly on cardiac myocytes, display adrenergic, cholinergic, and purinergic functions, sometimes in isolation but more often in combination. In this paper we describe additional effects on cardiac myocytes evoked by these neurons; the effects were excitatory and insensitive to adrenergic blocking agents (and to agents that block the inhibitory effects of acetylcholine and purines). In some of these microcultures, evidence consistent with secretion of serotonin was obtained; the nonadrenergic excitatory effect was diminished or abolished by serotonin blockers or reserpine. Further evidence for serotonergic transmission is presented in the accompanying paper by Sah and Matsumoto (1987). In other cases, an as-yet-unidentified agent "X" also produced a nonadrenergic excitation. The X effect characteristically required a prolonged train of neuronal impulses, had a time course of 50-200 sec, and was insensitive to agents that affected the other transmitters, including serotonin. In addition, we discuss 2 remarkable features of the transmitter repertoire of the microcultured sympathetic neurons: expression of the several transmitters in a variety of combinations, including at-least-quadruple function, and expression of the transmitters within a particular combination in varying relative strengths. The result is a diversity of transmitter release greater than that previously reported for vertebrate or invertebrate neurons.