Parasexual recombination was used to obtain improved chymosin-producing strains and to perform genetic analysis on existing strains. Chlorate resistance was used to select for a variety of spontaneous nitrate assimilation pathway mutations in strains previously improved for chymosin production using classical strain improved methods including mutation and screening, and selection for 2-deoxyglucose resistance (dgr). Diploids of these improved strains were generated via parasexual recombination and were isolated on selective media by complementation of nitrate assimilation mutations. A preliminary genetic analysis of diploid and haploid segregants indicated that the dgr trait, resulting in overexpression of chymosin, was recessive. Also, mutations in two different dgr genes resulted in an increased level of chymosin production. When these mutations were combined via parasexual recombination, the resulting haploid segregants produced about 15% more chymosin than either pal ental strain. CHEF gel electrophoresis was used to determine the chromosomal location of the integrated chymosin DNA sequences, and to verify diploidy in one case where the chromosome composition of two haploid parents differed.