Chromosomes from Cryptococcus neoformans, an encapsulated yeast pathogen, were separated by contour-clamped homogeneous field gel electrophoresis. Seven strains representing all four serotypes were studied. It was found that each strain had a unique, reproducible pattern of chromosome bands which could potentially be used for strain polymorphism studies. There were between 10 and 12 chromosomes in the strains studied, with an approximate genomic size of 15,000 to 17,000 kilobases. Chromosome separation also could be used to assign locations for cloned genes, and the ribosomal DNA genes were found on one of the larger C. neoformans chromosomes. The technique of electrophoretic karyotyping should be helpful for genetic and molecular investigations into the biology of C. neoformans.