AbstractNormal and reverse‐phase high‐performance liquid chromatography in conjunction with radioimmunoassay and mass spectrometry were used to identify the free and conjugated ecdysteroids (after enzymatic hydrolysis) from day‐4 pupae of the gypsy moth,Lymantria disparL. Seven ecdysteroids were searched for, but only 20‐hydroxyecdysone (964 ng/g fresh weight) and ecdysone (367 ng/g fresh weight) were detected. Analysis of conjugated ecdysteriods after liberation by hydrolysis also indicated the presence of 20‐hydroxyecdysone (21.6 ng/g fresh weight) and ecdysone (2.4 ng/g fresh weight). Neither 26‐hydroxyecdysone nor the 3α‐epimers of 20‐hydroxyecdysone or ecdyso