The penetration of the antifungal antibiotic - amphotericin B (AmB) -into Langmuir monolayers formed by cholesterol and ergosterol has been investigated by measuring the overpressure (Delta pi) in the film after the injection of AmB into water subphase. In addition, the penetration of AmB into sterol monolayers was studied at the microscopic level with Brewster angle microscopy (BAM) by recording BAM images simultaneously with the surface pressure increase. The obtained results show that monolayers formed by cholesterol are more penetrable for AmB molecules while rigid and closely packed films of ergosterol are less accessible for the antibiotic. This does not imply that the interactions between AmB and ergosterol are weaker than those between AmB and cholesterol, but indicates that the extent of AmB penetration is related to a different space accessibility. To get insight into the role of phospholipids in the interactions between AmB and sterols, the penetration of the antibiotic into mixed ergosterol-dipalmitoyl phosphatidylcholine (DPPC) and cholesterol-DPPC monolayers have also been investigated. The extent of AmB penetration into mixed monolayers was found to be dependent both on AmB bulk concentration and DPPC-sterol proportion in the mixed film. (c) 2004 Elsevier B.V. All rights reserved.