Ecdysteroid titres have been determined in adult female house crickets (Acheta domesticus) in relation to reproductive maturation. Ecdysteroid levels in newly emerged adult females are low except in the gut and carcass, which probably reflect the remnants of the preecdysial ecdysteroid peak. Ecdysteroid levels in all compartments increase markedly once ovarian weight surpasses 10 mg. Apolar ecdysteroid conjugates (ecdysone 22‐fatty acyl esters) predominate in ovarian tissue throughout ovarian maturation, but low levels of free ecdysteroid and polar conjugated ecdysteroids are also present. During this period, two peaks of ecdysteroids (mainly free and apolar conjugated ecdysteroids) are observed in the haemolymph, gut, and carcass compartments. The peaks in the haemolymph occur when the ovarian mass reaches 30 and 100 mg. The gut and carcass may be acting as sinks or sites of metabolism for the hormone released from the ovaries. The rate of ecdysone acylation by ovaries was found to be developmentally regulated, increasing from low levels in the immature ovaries of newly emerged females as the ovaries increase in size. A semiquantitative assay has been developed to identify compounds which inhibit the conversion of [3H] ecdysone into 22‐fatty acyl [3H] ecdysone by ovaries in vitro. A number of ecdysteroids possessing a free hydroxyl group at C‐22 as well as the side‐chain stereochemistry of ecdysone effectively inhibit this conversion, probably by acting as competitive substrates. In the cases of 20‐hydroxyecdysone and ponasterone A, it was clearly demonstrated that these compounds are converted to a mixture of C‐22 fatty acyl esters. Several other compounds which have been sugested to affect ecdysteroid metabolism/mode of action in other systems were also tested for their effects on the acyltransferase activity of ovaries in vitro. Arch. Insect Biochem. Physiol. 35:279‐299, 1997.© 1997 Wiley‐Liss, Inc.