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Serial isolates of Cryptococcus neoformans from 33 human immunodeficiency virus-infected patients with cryptococcosis were analyzed to determine whether persistence might result from reinfection with a new cryptococcal strain or acquisition of antifungal resistance. Isolates were subtyped by multilocus enzyme electrophoresis (MEE), electrophoretic karyotyping (EK), random-amplified polymorphic DNA (RAPD), and the CNRE-1 DNA probe, MICs of amphotericin B, fluconazole, and 5-fluorocytosine were determined. No changes in MEE or RAPD subtypes were detected in serial isolates from any patient. Isolates from 8 patients (24%) showed alterations in EK only (mobility change in two or more bands) but not with any other subtyping method. MICs did not change significantly in isolates from 30 patients. In 1 case, the fluconazole MIC increased stepwise over 18 months, suggesting development of resistance. These overall invariant subtyping and MIC results confirm previous studies suggesting that persistent cryptococcal infection is due to relapse rather than reinfection or antifungal drug resistance.