The HEM15 gene in Saccharomyces cerevisiae encodes ferrochelatase (EC 4.99.1.1, protoheme ferrolyase), a mitochondrial inner membrane-bound enzyme which catalyzes the insertion of ferrous ion into protoporphyrin IX, the last step in protoheme biosynthesis. The gene was isolated by functional complementation of a hem15 mutant. Sequence analysis of a 2.9-kilobase genomic DNA fragment revealed an open reading frame of 1179 nucleotides, plus a gene coding for a tRNA(Val)(GUU) and delta elements downstream from the 3'-end of HEM15. The open reading frame encodes a precursor form of the protein containing a 31-amino acid presequence. The mature enzyme contains 362 amino acid residues; its calculated molecular weight (40,900) and predicted amino-terminal sequence agree with those determined from the purified protein. It is relatively abundant in lysine (9%) and contains no apparent transmembrane segment. Disruption of the HEM15 gene led to non-viable cells in certain genetic background. Northern (RNA) analysis showed a slight (1.5-2-fold) repression of HEM15 expression by glucose.