Culturing cells on microcarriers maximizes the ratio of surface area to culture medium volume. The aim of this study was to show microcarrier culture as a useful tool for improvement of steroidogenic activity in mouse Leydig cells. Freshly isolated cells were grown on Sephadex microcarriers, Cytodex 3, and gelatin beads (gelaspheres) that were uncoated, coated with collagen or coated with fibronectin. The cells were cultured in control or LH-supplemented medium. The effect of LH on testosterone and estradiol secretion was studied with appropriate radioimmunoassays. The activity of hydroxysteroid dehydrogenases (3 beta-HSD, 17 beta-HSD) was evaluated histochemically. Leydig cells growing on microcarriers formed colonies. The strongest response to luteinizing hormone stimulation was observed on gelatin beads coated with fibronectin and collagen. In cells growing on a fibronectin layer, very strong activity of 3 beta-HSD and 17 beta-HSD and an increase in steroid hormone levels were observed. Basal and LH-stimulated testosterone and estradiol secretion were all much higher in the microcarrier than in the monolayer culture system. We conclude that Leydig cells maintain differentiated functions more efficiently on collagen- and fibronectin-coated gelasperes than on Sephadex microcarriers and that uncoated gelaspheres are less efficient than coated ones. Pure gelaspheres are unsuitable for improvement of the hormonal and enzymatic activity of Leydig cells.