Various waste frying oils (WFOs) were evaluated as substrates for rhamnolipid production by Pseudomonas aeruginosa mutant EBN-8 in the presence or absence of rhamnolipid precursor, under single-/batch-fed conditions. Soybean WFO was the best substrate, producing 9.3 g rhamnolipid l(-1) with the specific product yield of 2.7 g g(-1) h, under batch-fed cultivation with the addition of rhamnolipid precursor. The surface tension of the cell-free culture broth (CFCB) was 29.1 mN m(-1) and the interfacial tension against n-hexadecane was < 1 mN m(-1). The hydrocarbon/CFCB systems showed the relative emulsion stability to be in the range of 89.7-92.3.