Bovine NAD(+)-dependent isocitrate dehydrogenase was shown previously to contain four subunits of approx. 40 kDa (subunits 1-4) possessing different peptide maps and electrophoretic properties [Rushbrook and Harvey (1978) Biochemistry 17, 5339-5346]. In this study the heterogeneity is confirmed using enzyme purified by updated methods and from single animals, ruling out allelic variability. Subunits 1 and 2 were differentiated from each other and from subunits 3 and 4 by N-terminal amino acid sequencing. Subunits 3 and 4 (subunits 3/4) were identical in sequence over 30 residues. The N-terminal residues of subunits 1 and 2 were homologous but not identical with the beta- and gamma-subunits respectively of the comparable pig heart enzyme. Subunits 3/4 were identical over 30 residues with the N-terminus of the pig heart alpha-subunit. Full-length sequence, including that for mitochondrial import, is presented for a protein with the processed N-terminus of subunits 3/4, deduced from cloned cDNA obtained utilizing the N-terminal sequence information. The derived amino acid sequence for the mature protein contains 339 amino acids and has a molecular mass of 36,685 Da. Complete identity with N-terminal and Cys-containing peptides totalling 92 residues from the alpha-subunit of the pig heart enzyme [Huang and Colman (1990) Biochemistry 29, 8266-8273] suggests that maintenance of a particular three-dimensional structure in this subunit is crucial to the function of the enzyme. An electrophoretic heterogeneity within the pig heart alpha-subunit, similar to that shown by bovine subunits 3/4, was demonstrated. One reordering of the Cys-containing peptides of the pig heart alpha-subunit is indicated. Sequence comparison with the distantly related NADP(+)-dependent enzyme from Escherichia coli, for which the three-dimensional structure is known [Stoddard, Dean and Koshland (1993) Biochemistry 32, 9310-9316] shows strong conservation of residues binding isocitrate, Mg2+ and the NAD+ moiety of NADP+, consistent with a catalytic function.