The bacterial biosurfactants 3-[3'-(L-rhamnopyranosyloxy)decanoyloxy]decanoic acid (RL-1) and 3-[3'-(2"-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyloxy) decanoyloxy]decanoic acid (RL-2) were isolated from Pseudomonas aeruginosa DSM 2659 cultures. An HPLC method was developed for the p-bromophenacyl esters of the rhamnolipids. Separation was obtained within 25 min on a RP C18 column using a linear gradient of water-acetonitrile (30:70 to 0:100) and UV detection (265 and 320 nm). Linearity of response existed for 1.2-25 microg of the RL-1 and 1.8-25 microg of the RL-2-p-bromophenacyl ester (0.9-19.2 microg RL-1 and 1.3-18.0 microg RL-2). The reproducibility of the entire analytical method (extraction, derivatisation) was tested.