A process for producing rhamnolipids involving culturing Pseudomonas chlororaphis strain NRRL B-30761 in a first aqueous culture medium containing about 0.3% NH4H2PO4, about 0.2% K2HPO4, about 0.2% carbon source, about 0.5 mg/L FeSO4, and about 0.1% MgSO4 for about 24–about 48 hours at about 25°–about 30° C. with orbital shaking, and then culturing Pseudomonas chlororaphis strain NRRL B-30761 in a static second aqueous culture medium containing per liter about 2% carbon source, about 0.7 g KH2PO4, about 0.9 g Na2HPO4, about 2 g NaNO3, about 0.4 g MgSO4.7H2O, and about 0.1 g CaCl2.2H2O for at least about 72 hours at about 20°–about 23° C., wherein the first and second aqueous culture medium contains only one source of carbon.