Ecdysteroid levels were investigated by HPLC-MS over the moult cycle and in relation to reproduction in male shore crabs Carcinus maenas. Ecdysone (E), 20-hydroxyecdysone (20E) and Ponasterone A (PoA) were quantified in the haemolymph, hepatopancreas and testis. Also, the expression of 2 recently discovered Cytochrome p(450) genes (CYP330A1 and CYP4C39) inducible by ecdysteroids was studied in the hepatopancreas by Northern blot hybridisation analysis. In the haemolymph and hepatopancreas, all 3 ecdysteroids varied over the moult cycle with high levels in premoult and low levels in postmoult and intermoult. In the testis, 20E and E were present at high levels except in Postmoult Stage A, where low levels were observed. PoA was never observed in the testis. Ecdysteroids were quantified in the red and green colour forms of late intermoult C-4 crabs. In both phenotypes, 20E was the dominating ecdysteroid in late intermoult. In the haemolymph, 20E levels did not vary between the 2 phenotypes, but haemolymph 20E levels were negatively related to size. Also, haemolymph 20E levels varied with season in late-intermoult crabs, with higher levels during spring and autumn than during summer. Green crabs had significantly higher testicular E levels than red crabs. Ecdysteroid levels were negatively related to CYP330A1 and CYP4C39 gene expression. CYP330A1 and CYP4C39 mRNA levels were low during intermoult and premoult but high during postmoult. The results suggest that E and 20E are involved in both growth and reproduction whereas PoA is involved in moulting but not in reproduction and that the testis of male shore crab may be a possible source of ecdysone production in addition to the Y-organ. The results also support the concept that the probability of male shore crabs entering a new moult cycle decreases with increasing size.