Cytochrome b561 from bovine adrenal medulla chromaffin granules was purified in octylglucoside by hydrophobic chromatography. The apparent molecular weight was determined to be 30,000 by gel electrophoresis in sodium dodecyl sulfate. This molecular weight differs from that previously reported for cytochrome b561 purified by other procedures. The granule cytochrome was compared to mitochondrial cytochrome b and, in spite of similar molecular weights and optical spectra, these two proteins were found to be different by the following criteria: 1) amino acid composition, 2) NH2-terminal analysis, 3) interaction with antimycin, and 4) reduction with durohydroquinone.