This report describes experiments designed to demonstrate the suitability of the fission yeast Schizosaccharomyces pombe as a host for antisense RNA regulation. A lacZ gene-expressing yeast strain was constructed and used as a host for the expression of a series of antisense RNAs complementary to various regions of the target lacZ mRNA. All lacZ antisense genes were placed under control of the thiamine-repressible nmt1 promoter of S. pombe and expressed from episomal plasmids. For each antisense plasmid a corresponding sense control plasmid was constructed. All lacZ antisense genes were shown to express antisense RNAs of the expected size at equivalent steady-state levels. beta-Galactosidase activity in transformed cells expressing the long, short 5' or short 3' lacZ antisense RNAs was shown to be reduced by 45%, 20%, and 10%, respectively, relative to control transformants. Further experiments indicated that antisense RNA regulation in this system was conditional and reversible, with the observed reduction of beta-galactosidase activity being dependent on the transcription of lacZ antisense RNA. Our results represent the first successful example of antisense RNA regulation of gene expression in yeast and establish S. pombe as an experimental model for the biochemical analysis of antisense RNA regulation.