Previous studies utilizing relatively nonselective octopamine (OA) agonists have suggested that differences may exist between the pharmacological responses of Gs-linked OA receptors found in various insect tissues and species. The present experiments identify and characterize two structurally related phenyl(imino)imidazolidine (PII) derivatives that demonstrate a significant degree of OA receptor subtype selectivity and, when used in concert, are able to distinguish tissue- and/or species-related differences in OA receptor response. In membrane preparations, 2,4,6-triethyl-PII (TEP) showed > 60-fold selectivity for activating adenylate cyclase in the firefly light organ vs. that in Manduca sexta nerve cord, whereas 2,4,6-trimethyl-PII (TMP) showed > 2-fold selectivity for activating adenylate cyclase in Manduca nerve cord vs. that in the light organ. Within the same tissue, the potency ratio for TEP/TMP was 29 in the light organ and 0.2 in the nerve cord, a degree of selectivity much greater than that of other PII analogs examined. Corresponding physiological measurements of octopaminergic response correlated well with biochemical measurements. TEP was 24-fold more potent than TMP for OA-mediated light emission in intact firefly light organs, whereas TMP was more than 3-fold more potent than TEP as an antifeeding agent in Manduca. These results indicate that the PII analogs, TEP and TMP, should prove quite useful for characterizing and differentiating OA receptor subtypes in various tissues and species. In addition, these findings provide further evidence that light emission in fireflies and disruption of feeding in Manduca involve Gs-linked OA receptors, subtypes of which are distributed differently in the end organs of these two species.