Candida (Torulopsis) glabrata is an emerging nosocomial pathogen that may be relatively resistant to fluconazole. A series of 75 isolates (blood, urine, tissue, and other sites) from 16 patients (1 to 12 isolates per patient) at a single university medical center were analyzed by pulsed field gel electrophoresis (PFGE) of restriction endonuclease digests of chromosomal DNA. The MICs of the isolates for amphotericin B, flucytosine, fluconazole, and itraconazole, were determined by a microdilution broth method. A preliminary study of seven restriction enzymes, three producing small fragments (Hinf I, Hind III, Eco RI) and four producing large fragments (Eag I, BssH II, Sfi I, Not I) identified Not I as giving interpretable banding patterns. Isolates were considered of different types if they differed by two or more bands. Sixteen distinct DNA types (A to P) were identified. Karyotyping was used an an additional technique to compare strains with a common PFGE type. Most patients were colonized or infected with a single type at multiple body sites and over time. One PFGE type was shared by four patients housed in different areas of the institution at different times. For two of these four patients, the karyotype was also indistintuishable. Five patients were each colonized with two distinct types. The MIC of the strains studied were amphotericin B 0.5-1.0 microgram/ml (MIC90 = 1.0 microgram/ ml), 5-fluorocytosine 0.25-->256 micrograms/ml (MIC90 = 2 micrograms/ml), fluconazole 0.25-->128 micrograms/ml (MIC90 = 32 micrograms/ml), and itraconazole 0.06-8.0 micrograms/ml). Molecular typing by PFGE using Not I digestion is a useful technique for epidemiological investigation as epidemiologically related isolates are generally identical and epidemiologically unrelated isolates are different by this method.