With the final aim of identifying the testicular steroids involved in the feedback mechanism of the hypothalamus-pituitary-gonad axis, steroid secretion by the testis of African catfish, Clarias gariepinus, was studied in vitro, by means of gas chromatography followed by mass spectrometry. Testicular fragments of sexually mature catfish raised in captivity were incubated in L-15 medium with and without catfish pituitary extract (cfPE). Without adding cfPE, 22 steroids could be identified, amongst which 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone and 11-ketoandrostenedione were dominating. After incubation in the presence of cfPE, the concentrations of the four 11-oxygenated steroids were increased about 4-fold. The amounts of pregnane derivatives in the incubation medium showed the largest increases in the presence of cfPE. 5β-Pregnane-triol levels, for example, were 60-fold higher than in the medium from control incubations. The secretion of 5α- and 5β-androstanes was also stimulated by cfPE.The stimulation was not equal for all steroids, indicating that cfPE not only stimulates total steroidogenesis by increasing the availability of cholesterol, but also by influencing specific steroid converting enzyme activities.