To determine if major thymic neuropeptides and neurotransmitters can directly influence the functional activity of cultured rat thymic epithelium, neuropeptides and neurotransmitters were applied, and intercellular communication, proliferation, and thymulin secretion assessed. After injections of a mixture of lucifer yellow dextran (too large to pass gap junctions) and cascade blue (which does) into single cells, some neuropeptides decrease dye coupling: 0.1 mM GABA (P < 0.0001), 100 nM NPY (P < 0.0001), 100 nM VIP (P < 0.001), 100 nM CGRP (P < 0.001), 100 nM SP (P < 0.01), and 0.1 mM histamine (P < 0.01), whereas 0.1 mM 5-HT, 1 mM acetylcholine, and 1 microM isoproterenol (beta-adrenergic agonist) had no effect. Proliferation (incorporation of tritiated thymidine) was increased by CGRP (P = 0.004) and histamine (P < 0.02), but decreased by isoproterenol (P = 0.002), 5-HT (P = 0.003), and acetylcholine (P < 0.05). The percentage of multinucleate cells was decreased after isoproterenol (2.5%), and increased after 5-HT (21.3%), GABA (15%), and histamine (15.1%). Compared to controls, thymulin in the supernatant was decreased after challenge with acetylcholine (52%), isoproterenol (71%), 5-HT (73%), and histamine (84%). This study demonstrates direct effects of neuropeptides and neurotransmitters on functional aspects of cultured thymic epithelial cells.