As a prerequisite to standardization of dermatophyte susceptibility testing, conditions that support optimal growth of different dermatophyte species must be established. Eighteen isolates of Trichophyton spp. (T rubrum, T mentagrophytes, T tonsurans) were grown in 4 different media: RPMI 1640 with L-glutamine, without sodium bicarbonate and buffered at pH = 7.0; antibiotic medium #3 (Penassay); yeast nitrogen base with 0.5% dextrose buffered at pH = 7.0; and Sabouraud dextrose broth. Incubation for 6 days at 35° C produced the following results: RPMI and Sabouraud dextrose supported equally sufficient growth for all strains tested; Penassay supported growth of only 33% of the isolates tested, and buffered yeast nitrogen base did not support growth of any isolates. RPMI was selected as the optimal medium, and organisms were tested at both 30° C and 35° C with a standardized inoculum density of 10³ conidia/mL. No temperature differences were noted in the amount of growth of the dermatophytes tested. With RPMI at an incubation temperature of 35° C, 3 inoculum sizes (10³, 10⁴, and l0⁵ conidia/mL) were tested against 4 antifungal agents: griseofulvin, itraconazole, terbinafine, and fluconazole. Inoculum size did not affect minimum inhibitory concentration (MIC) results for itraconazole or terbinafine, but a larger inoculum produced a slightly higher MIC for griseofulvin and a noticeably higher MIC for fluconazole. Our data support the use of RPMI 1640, 35° C, and 4 days as an incubation temperature and time, respectively, and an inoculum of 10³ conidia/mL as optimal conditions for the determination of the antifungal susceptibility of dermatophytes. (J Am Acad Dermatol 1999;40:S9-13.)