Pseudomonas aeruginosa strain PA01, which is normally sensitive to 10 units/ml of polymyxin, was adapted in eight successive steps to be resistant to polymyxin at 6000 units/ml. The polymyxin-resistant variant was very sensitive to all other antibiotics with which it was challenged, including hydrophobic antibiotics such as erythromycin. This increased sensitivity implies that the acquired polymyxin resistance had disrupted the permeability barrier of the outer membrane. Changes in the outer membrane protein profile of PA01 were studied at each adaption step using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At the third step (resistant to 80 units/ml) protein H1 was absent. At succeeding steps up to 6000 units/ml of polymyxin, protein H1 was still absent and the levels of OM proteins D, E, F (major porin) and G were all considerably reduced compared with the wild type. Overproduction of protein H1 by wild type P. aeruginosa under conditions of magnesium depletion has been proposed as a mechanism to explain polymyxin resistance. However, protein H1 remained absent from the outer membrane of the polymyxin-resistant variants even under magnesium depletion. Furthermore, protein H1 could not be induced by magnesium depletion when the resistant variant was repeatedly grown in concentrations of polymyxin as low as 10 units/ml yet, at this low concentration, the variant retained its resistance to 6000 units/ml. These observations make it unlikely that induction of protein H1 alone is a mechanism of polymyxin resistance in P. aeruginosa.