Abstract The O-pentafluorobenzyloxime (OPFB)-heptafluorobutyrylester (HFB) derivatives of hemolymph steroids extracts from both male and female Astacus leptodactylus were subjected to negative ion chemical ionization and capillary gas chromatography-mass spectrometry (NCI/GC-MS). Five nonecdysteroid steroids, namely pregnenolone, 17α-hydroxypregnenolone, testosterone, cholesterol, and 6β-hydroxyprogesterone were identified. With selected ion monitoring (SIM), indications were found for the presence of four more steroids: androstenedione, 5α-dihydrostosterone, 11-ketotestosterone, and 11β-hydroxytestosterone. The presence of 6β-hydroxyprogesterone could only be demonstrated in the female hemolymph. With the technique used, dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, and estrogens could not be detected in male and female hemolymph extracts.