The electrophoretic karyotypes of 32 clinical and 3 environmental Cryptococcus neoformans isolates from New York City were studied by contour clamped homogeneous electrophoresis. There was extensive variation among the electrophoretic karyotypes of isolates from different patients. Sequential C. neoformans isolates from patients with chronic or relapsing infection had very similar karyotypes. However, minor differences in electrophoretic karyotypes were detected among sequential isolates from 50% of the patients studied, suggesting the occurrence of chromosomal rearrangements or deletions in vivo. This hypothesis was tested by infecting mice, recovering isolates from mouse organs, and comparing the electrophoretic karyotypes before and after passage. Three clinical and three environmental strains were studied before and after passage in mice. Karyotype differences were detected after mouse passage for one clinical and two environmental strains. Our results indicate (i) extensive karyotype variation among isolates from a small geographic regions, (ii) a high frequency of electrophoretic karyotype differences among sequential isolates from individual patients, and (iii) the occurrence of electrophoretic karyotype changes during experimental infection of mice. The implications of these observations are discussed.