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Since the nucleotide sequence of bovine adrenodoxin cDNA is at variance with protein sequencing data in that it encodes an additional 14 amino acids at the COOH terminus, we used a specific antibody raised against this 14-amino-acid segment to examine its presence in: nascent precursor polypeptide chains, the processed mature adrenodoxin and mitochondria of both steroidogenic and nonsteroidogenic tissues. These studies reveal the presence of the extra peptide in the precursor form derived from in vitro translation and in the newly synthesized mature form as shown by [35S]methionine labeling of proteins in adrenocortical cells. Both the purified COOH-terminal synthetic peptide and purified mature adrenodoxin competed with radiolabeled adrenodoxin for immunoprecipitation by the anti-peptide antibody. Immunoblots revealed the presence of the extra peptide in purified adrenodoxin and in bovine adrenocortical, corpus luteal, kidney and liver mitochondria while it was not detectable in heart mitochondria. Thus, we conclude that mature adrenodoxin and its homologs in non-steroidogenic tissues contain the C-terminal extension following uptake into mitochondria. These results indicate structural homology between adrenodoxin and the iron-sulfur proteins of the kidney and liver and also suggest the presence of a second iron-sulfur protein in kidney and liver.