Using plasmids containing the genes for thymidine kinase (tk) and neomycin resistance (neo), we have shown that DNA-mediated genotypic transformation of L and Chinese hamster ovary (CHO) cells is increased several-fold by the presence of the sterol-binding polyene antibiotic, amphotericin. Transformation into the same host cells, using genomic DNA, was also enhanced by amphotericin. Phenotypic expression of beta-galactosidase activity of a plasmid containing the gene for the enzyme was also markedly elevated when the antibiotic was added at transfection. Other sterol-binding polyene antibiotics also showed activity in these DNA-mediated gene transfer assays.