AbstractTestes from late last stage larvae of the tobacco budworm,Heliothis virescens, were incubated with [3H]ecdysone and [3H]cholesterol. [3H]Ecdysone was converted to six other major ecdysteroids, identified by cochromatography in reverse‐phase high‐pressure liquid chromatography (RPHPLC); four of them were verified by normal‐phase HPLC. A highly polar fraction, moderately polar ecdysteroids (20,26‐dihydroxyecdysone, 3‐epi‐20‐hydroxyecdysone, and 20‐hydroxyecdysone) and low‐polarity ecdysteroids, including 2‐deoxyecdysone, were detected after incubation with [3H]ecdysone. Compounds that reacted positively to antibodies to progesterone and testosterone were detected in the low‐polarity fractions. Testes were incubated in fractions corresponding to each of the major ecdysteroid peaks derived from [3H]ecdysone metabolism. Although most of the radioactive ecdysteroid fractions were further metabolized to high‐ and low‐polarity endpoints, 88% of the [3H]20‐hydroxyecdysone peak apparently remained unmetabolized. 20‐Hydroxyecdysone may be the primary ecdysteroid product of testes ofH. virescens.[3H]Cholesterol was not metaboli