The effects of serotonin [5-hydroxytryptamine (5-HT)] on the transepithelial electrical properties of the short-circuited rabbit conjunctiva were examined. With this epithelium, the short-circuit current (I_sc) measures Cl secretion plus an amiloride-resistant Na⁺ absorptive process. Apical addition of 5-HT (10 µM) elicited a prompt I_sc reduction from 14.2 ± 1.2 to 10.9 ± 1.2 µA/cm² and increased transepithelial resistance from 0.89 ± 0.05 to 1.03 ± 0.06 k · cm² (means ± SE, n = 21, P < 0.05). Similar changes were obtained with conjunctivae bathed without Na⁺ in the apical bath, as well as with conjunctivae preexposed to bumetanide with the Cl-dependent I_sc sustained by the parallel activities of basolateral Na⁺/H⁺ and Cl/HCO exchangers. In contrast, the 5-HT-evoked effects were attenuated by the absence of Cl (I_sc = 0.5 ± 0.2, n = 5), suggesting that reduced Cl conductance(s) is an effect of 5-HT exposure. In amphotericin B-treated conjunctiva and in the presence of a transepithelial K⁺ gradient, 5-HT addition reduced K⁺ diffusion across the preparation by 13% and increased transepithelial resistance by 4% (n = 6, P < 0.05), indicating that an inhibition in K⁺ conductance(s) was also detectable. Significant electrical responses also occurred under physiological conditions when 5-HT was introduced to epithelia pretreated with adrenergic agonists or protein kinase C, phospholipase C, phosphodiesterase, or adenylyl cyclase inhibitors or after perturbation of Ca²⁺ homeostasis. Briefly, the conjunctiva harbors the only known Cl-secreting epithelium in which 5-HT evokes Cl transport inhibition; receptor subtype and signal transduction mechanism were not determined.