Cytochrome c has been purified to homogeneity from alligator liver (Alligator mississipiensis) using aluminum sulfate precipitation, CM-cellulose and gel-filtration chromatography, and reverse-phase HPLC. The protein exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an approximate molecular weight of 12,000 Da. Oxidized and reduced visible spectra yielded maxima at 408 (tau) nm and 315 (delta), 415 (tau), 520 (beta), and 550 (alpha) nm, respectively, while potentiometric titrations in the presence of dye-mediators yielded an Eo of +265 mV. N-terminal analysis of the protein yielded no sequence which indicates a blocked residue. A combination of amino acid sequencing, using peptides obtained from Staphylococcus aureus V8 protease, endoproteinase Lys-C, and CNBr digests of the protein and total amino acid analyses, using equine and avian cytochromes c as standards yielded the primary sequence GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLIGRKTGQAPGFSYTEANKNKGITWGEETLMEYLE NPKKYIPGTKMIFAGIKKKPERADLIAYLKEATSN. Comparison with sequences of other cytochromes c indicated the closest similarity to cytochrome c from snapping turtle (Chelydra serpentina) with substitutions at five positions corresponding to residues 32 (His-->Asn), 44 (Glu-->Pro), 89 (Ala-->Pro), 100 (Asp-->Glu), and 104 (Lys-->Asn), respectively. The presence of Pro and Asn residues at positions 89 and 104, respectively, are unique to alligator cytochrome c.