Three new antisera have been produced in rabbits immunized with E-2/3-hemisuccinate conjugates. In a competitive enzyme immunoassay (EIA) with 20-ecdysone-peroxidase as a tracer, the antisera exhibit high specificity towards the side chain of the ecdysone (E) molecule. Modifications of the A-ring do result in no or small changes of sensitivity. One antiserum (RUD-2) allows the direct quantification of total ecdysteroids in samples containing E and 3-dehydroecdysone in varying ratios, without further sophisticated sample treatment. At least one of the other antisera allows the direct quantification of mixtures of E and 25-deoxyecdysone (RUD-1). Thus, these antisera may be a useful tool in studying molting control in arthropods.