The biosynthesis of gentamicin (GM) in shake flask cultivation and a resting cell system were studied. Suitable amounts of NaNO3, MgSO4, NaCl, penicillin, fosfomycin, cephradine, alpha-ketoglutarate, biotin and Tween 20 favoured the biosynthesis of GM. The ingredients of the optimum synthetic medium for the resting cell system are given. The amount of GM adsorbed on 1 g of wet mycelia and wet cell wall was 4775 and 20 000 U, respectively. Usually only 20-30% of the GM is excreted into the broth during fermentation, whereas about 75% of the bound GM can be released by ultrasonic treatment. The production of GM increased by 70% when 0.8 ml of mycelia (GM depleted) were added into 25 ml of fermentation broth at 90 h after inoculation. (C) 2002 Elsevier Science Ltd. All rights reserved.