Amphotericin B is a polyene macrolide antibiotic which interacts specifically with sterols in mammalian cell membranes. Amphotericin B-resistant (AMBr) lines of stable phenotype have been isolated from cultured Chinese hamster (V79) cells. Three AMBr clones (AMBr-1, -2 and -3) isolated independently after treatment with nitrosoguanidine were resistant to greater than or equal to microgram/ml of the antibiotic, while DNA synthesis as well as the colony-forming ability of the parental V79 cells was blocked by greater than 80% of control in the presence of 20--50 microgram/ml amphotericin B. The AMBr cell line also exhibited increased resistance to other polyene macrolide antibiotics such as nystatin and pentamycin. Other agents, however, such as cytosine arabinoside or ricin, blocked DNA synthesis in AMBr cells to the same extent as in V79 cells. The amphotericin B resistance phenotype was stably retained even after AMBr cells were cultured in the absence of the drug for over 200 generations. The control of free cholesterol or its esters was significantly decreased in all three resistant clones. Furthermore, cholesterol synthesis from acetate as well as mevalonate was partly defective in AMBr cells, compared with that in V79 cells.