In embryos, larvae, and juveniles of Pycnogonum litorale, unusual ecdysteroids have been found at excessively high levels. Concentrations and dynamics of the following eight ecdysteroids were determined by HPLC: 20-hydroxyecdysone, 20-hydroxyecdysone 22-glycolate, (25R) and (25S) isomers of 20,26-dihydroxyecdysone 22-acetate, 22-deoxy-20,26-dihydroxyecdysone, 20-hydroxyecdysone 22-acetate, 22-deoxy-20-hydroxyecdysone, and ecdysone 22-glycolate. The excessively high content of ecdysteroids is found in all developmental stages. Juvenile pycnogonids contain the highest total ecdysteroid amounts ever found in arthropods: 0.1% of their body dry weight. 20-Hydroxyecdysone 22-acetate is the prominent ecdysteroid and represents at all stages of both sexes, 66.2-85.8% of the total ecdysteroids. This compound, as well as all other ecdysteroids without a free 22-OH group, are presumed to be inactive as molting hormones. In contrast to these compounds, 20-hydroxyecdysone is present only in amounts similar to those in other arthropods. Furthermore, it is the only ecdysteroid with its peak at apolysis. It is regarded as the molt-promoting hormone. The origin and function of the other ecdysteroids in different developmental stages are discussed.