For development of a homologous transformation system for the zygomycete fungus, Rhizomucor pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R. pusillus IFO 4578 by the DNA-probing method with the leuA sequence of Mucor circinelloides as probe. The nucleotide sequence revealed that leuA of R, pusillus encoded a 755-amino-acid protein of 82.5 kDa with no intron. The leuA gene on pUC19 (plasmid pRPLeu10) was introduced by polyethyleneglycol-assisted transformation into protoplasts of a leuA(-) mutant of R, pusillus that was obtained by UV mutagenesis. Transformation under optimal conditions yielded 20 Leu(+) transformants (mu g pRPLeu10 DNA)(-1) (1 x 10(6) viable protoplasts)(-1) Blot analysis of DNA from the transformants Showed that the pRPLeu10 sequence was integrated into the genome by homologous recombination at the leuA locus.