A minichromosome of approximately 270 kilobases was generated following complementation of a ura5 mutant strain of C. neoformans with the plasmid pURA5g2. This is the first report of the in-vivo generation of a minichromosome by the method of electroporative transformation. The minichromosome occurred at a relatively high (> 20%) frequency in transformants that were stable for uracil protoprophy. The minichromosome was maintained in linear form as a large extrachromosomal element of the normal karyotype. Gel-purified DNA from the minichromosome readily transformed the ura5 mutant of C. neoformans. Southern-blot analysis of the minichromosome revealed the presence of multiple copies of the URA5 gene and ribosomal DNA sequences in addition to containing telomere-like sequence repeats. The minichromosome was transmitted through mitosis and meiosis with extremely-high fidelity.