The sequence of a cloned copy of the yeast ADE3 gene, which encodes the trifunctional enzyme C1-5,6,7,8-tetrahydrofolate (THF) synthase, was determined. Yeast cells transformed with a multicopy yeast plasmid containing this ADE3 gene overexpress C1-THF synthase 20-60-fold relative to wild-type yeast cells. C1-THF synthase from transformed cells is identical with that isolated from wild-type cells by all the criteria tested. The translated DNA sequence and amino-terminal protein sequences are identical and the amino acid composition predicted from the DNA sequence agrees closely with that determined by hydrolysis of C1-THF synthase protein. Correlation of the genetic map of the ADE3 region and of proteolysis experiments with the protein sequence suggests locations for two functional domains within yeast C1-THF synthase. The sequence of C1-THF synthase does not appear to be homologous to any other sequenced protein, including other proteins that use folate substrates. The 5' and 3' untranslated regions of the ADE3 gene suggest initiation and termination signals similar to transcription signals associated with other yeast genes. No special regulatory features have been associated with the ADE3 sequence. An unusual open reading frame that is encoded by a very unbiased set of codons follows the ADE3 gene.