Expression of the URA3 gene of Saccharomyces cerevisiae was studied by analysis of URA3-lacZ gene fusions constructed in vitro. Synthesis of hybrid beta-galactosidase by fusions in frame with the coding sequence for orotidine-5'-phosphate decarboxylase (OMPdecarboxylase) was found to be normally regulated even when only 11 nucleotides of URA3 coding sequence remained, indicating that all transcription initiation and regulatory sites are present at the beginning of the URA3 gene. An upstream initiator codon that begins a short overlapping coding sequence in another reading frame was also found to be active in producing hybrid beta-galactosidase. However this beta-galactosidase synthesis showed little or no regulation. Nuclease protection experiments revealed numerous species of URA3 mRNA. The regulation of these is consistent with the idea that the URA3 protein and the overlapping peptide are translated from differentially regulated mRNAs of different lengths.