Optimization of the medium for biosurfactants production by probiotic bacteria (Lactococcus lactis 53 and Streptococcus thermophilus A) was carried out using response surface methodology. Both biosurfactants were proved to be growth-associated, thus the desired response selected for the optimization was the biomass concentration. The selected factors based on MRS medium for L. lactis 53 growth were peptone, meat extract, yeast extract, lactose, ammonium citrate and KH2PO4. For S. thermophilus A based on the M17 medium, the selected factors were peptone, meat extract, yeast extract, lactose, soya peptone and sodium glycerophosphate. The optimum MRS composition was found to be 38.6 g/l peptone, 43.0 g/l lactose, 10 g/l. meat extract, 5.g/l yeast extract, 1.08 g/l Tween-80(E), 2 g/l KH2PO4, 2 g/l CH3COONa, 2 g/l ammonium citrate, 0.2 g/l MgSO(4)center dot 7H(2)O and 0.05 g/l MnSO(4)center dot 4H(2)O. The optimized medium allowed a mass of produced biosurfactant (milligram per gram cell dry weight) 1.6 times higher compared to MRS standard medium. The optimum medium composition for growing S. thermophilus A consists of 5.0 g/l peptone, 5.7 g/l lactose, 5.0 g/l meat extract, 2.5 g/l yeast extract, 5.0 g/l soya peptone, 26.4 g/l sodium glycerophosphate, 0.5 g/l ascorbic acid and 0.25 g/l MgSO(4)center dot 7H(2)O. With the optimization procedure a biosurfactant mass recovery 2.1 times higher was achieved. The application of response surface methodology resulted in an enhancement in biosurfactants production. (C) 2005 Elsevier Ltd. All rights reserved.