Pseudomonas aeruginosa AT10 produced a mixture of surface-active rhamnolipids when cultivated on mineral medium with waste free fatty acids as carbon source. The development of the production process to an industrial scale included the design of the culture medium. A 2(4) full factorial, central composite rotational design and response surface modelling method (RSM) was used to enhance rhamnolipid production by Pseudomonas aeruginosa AT10. The components that are critical for the process medium were the carbon source, the nitrogen source (NaNO3), the phosphate content (K2HPO4/KH2PO4 2:1) and the iron content (FeSO4.7H(2)O). Two responses were measured, biomass and rhamnolipid production. The maximum biomass obtained was 12.06 g dm(-3) DCW, when the medium contained 50 g dm(-3) carbon source, 9 g dm(-3) NaNO3, 7 g dm(-3) phosphate and 13.7 mg dm(-3) FeSO4.7H(2)O. The maximum concentration of rhamnolipid, 18.7g dm(-3), was attained in medium that contained 50 g dm(-3) carbon source, 4.6 g dm(-3) NaNO3, 1 g dm(-3) phosphate and 7.4 mg dm(-3) FeSO4.7H(2)O.