Candida lusitaniae is an important nosocomial pathogen that may express resistance to one or more antifungal agents including amphotericin B. We investigated the genotypic diversity and antifungal susceptibility among 47 clinical isolates from 33 patients hospitalized in 12 different medical centers. Strain delineation was performed by restriction endonuclease analysis of genomic DNA (REAG) with the restriction enzyme Sfi I followed by pulsed-field gel electrophoresis and by electrophoretic karyotyping (EK). Antifungal susceptibility of the isolates to amphotericin B, 5-fluorocytosine (5FC), fluconazole, and itraconazole was determined using National Committee for Clinical Laboratory Standards standard methods. Minimum inhibitory concentration (MIC)90 values ranged from 0.5 micrograms/ml for itraconazole to 512 micrograms/ml for 5FC. In general, isolates from a given patient or epidemiologically related isolates from a nosocomial cluster were identical by molecular typing methods. Epidemiologically unrelated isolates were all distinctly different by both EK and REAG typing methods. Although elevated amphotericin B MICs ( > or = 2 micrograms/ml) were observed in only three isolates, extended incubation (72 h) revealed amphotericin B MICs of 2-16 micrograms/ml in a subset of isolates suggesting potential amphotericin B resistance. These data document the genetic diversity, nosocomial transmission, and antifungal susceptibility of clinical isolates of C. lusitaniae.