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The major ecdysteroids of last-stage Oncopeltus fasciatus larvae were determined by HPLC/RIA analysis. An ecdysteroid other than ecdysone or 20-hydroxyecdysone, probably makisterone A, was the major ecdysteroid in hemolymph and in whole body extracts of 4- to 5-day-old larvae, when hemolymph ecdysteroid titers were at their peak. Little, if any, ecdysone or 20-hydroxyecdysone was detected in the hemolymph. Bioassay of the relative activities of ecdysone, 20-hydroxyecdysone, and makisterone A in last-stage larvae revealed that makisterone A was ten times more active than 20-hydroxyecdysone, and that ecdysone was inactive at doses as high as 1 mg/ml, its limit of solubility in 10% ethanol. Thus, it is suggested that makisterone A is the molting hormone in Oncopeltus. The implications of this discovery are discussed.