Y-organs are paired glands in crustaceans that secrete a class of steroid hormones (ecdysteroids) that regulate growth, molting and development. The glandular secretion has been assumed to be solely the ecdysteroid, ecdysone, a polyhydroxylated derivative of cholesterol. We previously reported that Y-organs of a crab (Cancer antennarius) additionally secreted an ecdysteroid that is less polar than ecdysone. Evidence is presented here that the other secretion product is 3-dehydroecdysone (3-dhE). The compound co-chromatographed with authentic 3-dhE in both normal-phase, and reversed-phase, high-performance liquid chromatography. Mass spectrometry of the ecdysteroid gave results consistent with its identity as 3-dhE. The putative 3-dhE was radiolabeled by injecting crabs with [3H]cholesterol and then incubating the Y-organs. The putative [3H]3-dhE secretion was then subjected to chemical reduction. The reaction yielded labeled products that co-chromatographed with authentic ecdysone and 3-epiecdysone. Results of other experiments gave the following results: (1) Putative 3-dhE was not altered (Chromatographie criteria) by incubations with snail hydrolases. (2) Putative [3H]3-dhE, added to incubations of Y-organ halves or homogenates, was not significantly converted to ecdysone; also, no conversion was evident after incubation in medium alone in which the hemolymph serum supplement was raised to 50% of the volume. (3) [3H]Ecdysone was not converted to putative 3-dhE in vitro by Y-organ halves or homogenates. These results support the conclusions that (a) Y-organs of C. antennarius secrete 3-dhE as well as ecdysone, (b) 3-dhE is the dominant secretion, and (c) the two ecdysteroids are synthesized in Y-organs from a common sterol precursor by separate final metabolic pathways. © 1989.