The in vitro activity of 1-[(4-Biphenylyl)-phenylmethyl]-1H-imidazole bifonazole, Bay h 4502, Mycospor) a new antifungal imidazole derivative, against a variety of medically important fungi were studied using an agar dilution procedure. Two imidazole drugs which have already appeared on the market as topical antimycotics, clotrimazole and miconazole, were also tested as reference drugs. MIC values of bifonazole to a range of pathogenic fungi and yeasts ranged from 0.04 to 80 micrograms/ml, but mostly were less than 2.5 micrograms/ml. The antifungal potency of bifonazole relative to that of clotrimazole and miconazole varied from strains to strains or from species to species. For example, almost all strains of dermatophytes were less susceptible to bifonazole than clotrimazole and the least susceptible to miconazole. Against most other fungi tested bifonazole was similar to or greater than clotrimazole and/or miconazole in inhibitory activity. The activity against Candida species increased in the order of bifonazole, miconazole and clotrimazole. When tested against other pathogenic yeasts such as T. glabrata and C. neoformans, bifonazole was more active than clotrimazole, and compared favorably with miconazole. Bifonazole and the other two imidazole derivatives inhibited the growth of most fungi at concentrations well below their quoted MICs. Composition and pH of culture medium, as well as fungal inoculum size, significantly altered the activity of bifonazole. A brief discussion of the possible clinical usefulness of bifonazole is given.