The pharmacology of prejunctional serotonin (5-HT) heteroreceptors that regulate the release of norepinephrine (NE) was studied in isolated bovine and human iris-ciliary bodies. The effect of exogenous 5-HT and various 5-HT receptor agonists was examined on the release of [3H]-norepinephrine ([3H]NE). Both 5-HT and m-chlorophenyl-biguanide (m-CPBG) caused enhancement in the field-stimulated release of [3H]NE from bovine tissues whereas 5-carboxamidotryptamine (5-CT) had no such effect. On the other hand, 8hydroxy-dipropylaminotetralin (8-OH-DPAT), caused a significant dose-related inhibition of evoked [3H]NE release. In human iris-ciliary bodies, 5-HT caused an inhibitory response on electrically-evoked [3H]NE release at low concentrations but produced an excitatory action at concentrations greater than 3 microM. To further confirm the nature of the prejunctional 5-HT heteroreceptors regulating [3H]NE release, effects of 5-HT3, 5-HT6 and 5-HT7 receptor antagonists were examined on a standard response to 5-HT. All antagonists examined caused a concentration-dependent inhibition of the response elicited by the standard 5-HT-induced response with the following rank order of potency (as measured by IC30 values): MDL-72222 >> SB-258719 > RO-04-690. We conclude that the excitatory prejunctional 5-HT heteroreceptors present in bovine iris-ciliary bodies belong to the 5-HT3 receptor subtype.