Despite their abundance and their enormous significance for various ecological processes, endocrine systems of microcrustaceans have been poorly investigated. Here, we used a highly sensitive radioimmunoassay (RIA) to determine free and conjugated ecdysteroid levels in whole-body extracts of adult Daphnia magna during a complete molt cycle. Ecdysteroid levels were predominated by free ecdysteroids. Starting from basal levels in the postmolt stage the concentration of free ecdysteroids increased sharply in the early premolt stage, followed by a sharp decline back to basal levels just prior to ecdysis. Polar and apolar ecdysteroid conjugates were found in rather low amounts with little variation during the molt cycle. Only small amounts of ecdysteroids were found in newly deposited eggs of D. magna, which suggest a sparing investment of maternal ecdysteroids into the eggs for early embryogenesis. As in whole-body extracts, free ecdysteroids were the predominant ecdysteroids found in eggs of D. magna, together with small amounts of polar and intermediary amounts of apolar conjugates. Hence, the pathways leading to polar and apolar ecdysteroid conjugates appear to be of minor importance in D. magna. Analyses of the immunodetectable peak in free ecdysteroids by liquid chromatography-mass spectrometry (LC-MS) revealed that molting is induced most probably by an increased level of 20-hydroxyecdysone. Microcrustaceans of the genus Daphnia are key components in freshwater food webs. Examination of the functional role of ecdysteroids in controlling developmental processes might help to understand the performance of the herbivorous grazer in its environment, in particular with regard to the adverse effects of environmental xenobiotics acting as endocrine disrupters.