Inhibition of Saccharomyces cerevisiae arginase (CAR1) gene expression was investigated using the antisense RNA technique. CAR1 DNA fragments containing the yeasty CAR1 gene sequences from the transcription initiation site (-49) or translation initiation site (+1) to the +501 region were amplified using PCR and inversely fused to the yeast CYC1 promoter on the yeast YIp5 plasmid. These recombinant plasmids were transformed into yeast cells to construct strains containing CYC1 promoter-antisense CAR1 DNA in their chromosomal DNA. When the CAR1 DNA region from -120 to +552 was amplified by PCR, the CYC1 promoter-antisense CAR1 DNA plasmid transformants produced the same size of PCR fragments,as vector only transformants, suggesting the recombinant plasmids did not integrate into the CAR1 loci. The level of arginase production by the recombinant transformants markedly decreased to about 15% of the enzyme activity produced by the vector only transformants.