Several temperature-sensitive cell division cycle (cdc) mutations differentially affect the regulatory step for cell proliferation in the yeast Saccharomyces cerevisiae. We recently found that one of these mutations, cdc63-1, resides in a gene called PRT1; other mutations in this gene had been previously shown to affect translation initiation. Here we report the molecular cloning and characterization of the PRT1 gene from yeast. Our results show that the PRT1 gene is an essential, single-copy gene which encodes a 2500-nucleotide polyadenylated transcript. The nucleotide sequence indicates that the gene could code for a protein product of Mr 88,000, which bears no overall amino acid sequence similarity to any other known protein but which contains similarity over a limited region to amino acid sequences involved in nucleotide binding.