We investigated the in vitro activity of nystatin and liposomal nystatin against 103 Candida isolates to determine the effect of both time and medium on MICs. We also compared the nystatin MICs with those of amphotericin B and fluconazole. Testing was performed in accordance with the National Committee for Clinical Laboratory Standards M27-A microdilution methodology with RPMI 1640, RPMI 1640 supplemented with glucose to 2% (RPMI-2), and antibiotic medium 3 supplemented with glucose to 2% (AM3). While nystatin MICs were similar to or slightly lower than liposomal nystatin MICs in RPMI 1640 and RPMI-2, they were markedly higher than liposomal nystatin MICs in AM3. Use of AM3 and determination of the MIC after 24 h of incubation provided a slightly wider range of liposomal nystatin MICs (0.06 to >16 mug/ml). Under these conditions, the MICs at which 90% of isolates were inhibited of nystatin and liposomal nystatin were 2 and I mug/ml, respectively. Nystatin and liposomal nystatin in general showed good activity against all Candida spp. tested. Although the MICs of nystatin and liposomal nystatin tended to rise in parallel with the amphotericin B MICs, nystatin and liposomal nystatin MICs of I to 2 and 0.5 to I mug/ml, respectively, were obtained for seven and six, respectively, of nine isolates for which amphotericin B MICs were greater than or equal to0.25 mug/ml. No correlation between fluconazole and nystatin or liposomal nystatin MICs was observed. As amphotericin B MICs of greater than or equal to0.25 mug/ml correlate with in vitro resistance, these results suggest that liposomal nystatin might have activity against some amphotericin B-resistant isolates. In vivo testing in animal models is required for clarification of this issue.