A full-length cDNA encoding porcine heart aconitase was derived from lambda gt10 recombinant clones and by amplification of the 5' end of the mRNA. The 2700-base pair (bp) cDNA contains a 29-bp 5' untranslated region, a 2343-bp coding segment, and a 327-bp 3' untranslated region. The porcine heart enzyme is synthesized as a precursor containing a mitochondrial targeting sequence of 27 amino acid residues which is cleaved to yield a mature enzyme of 754 amino acids, Mr = 82,754, having a blocked amino terminus. The NH2-terminal pyroglutamyl residue of the mature enzyme was identified by fast atom bombardment mass spectrometry and sequence analyses of an NH2-terminal peptide. Mature porcine heart aconitase contains 12 cysteine residues. Cysteines 358, 421, and 424 are ligands to the Fe-S cluster in the inactive [3Fe-4S] (Robbins, A. H., and Stout, C. D. (1989) Proteins 5, 289-312) and active [4Fe-4S] (Robbins, A. H., and Stout, C. D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3639-3643) forms. An alignment of the derived porcine heart sequence with 8 cysteine-containing tryptic peptides from bovine heart aconitase (Plant, D. W., and Howard, J. B. (1988) J. Biol. Chem. 263, 8184-8189; Plank, D. W., Kennedy, M. C., Beinert, H., and Howard, J. B. (1989) J. Biol. Chem. 264, 20385-20393) shows that 198 of 202 amino acids are conserved and suggests that the two enzymes are virtually identical.