The Pseudomonas aeruginosa PA1 strain, isolated from the water of oil production in Sergipe, Northeast Brazil, was evaluated as a potential rhamnolipid type of biosurfactant producer. The production of biosurfactants was investigated using different carbon sources (M-hexadecane, paraffin oil, glycerol, and babassu oil) and inoculum concentrations (0.0016-0.008 g/L). The best results were obtained with glycerol as the substrate and an initial cell concentration of 0.004 g/L. A C:N ratio of 22.8 led to the greatest production of rhamnolipids (1700 mg/L) and efficiency (1.18 g of rhamnolipid/g of dry wt).