A heme-not-containing protein was isolated from cytochrome c1 preparation by gel filtration after carboxymethylation and citraconylation. The amino acid sequence of this protein was determined by the analysis of tryptic and chymotryptic peptides as well as by solid-phase sequence analysis. It consisted of 78 amino acid residues and the molecular weight was calculated to be 9,175. This protein contained a high proportion of glutamic acid and glutamine (27% of the total residues) but no methionine, isoleucine, tyrosine, and tryptophan. The most notable feature was an acidic cluster of 8 consecutive glutamic acid residues near the amino(N)-terminus). The secondary structure was predicted to have a high proportion of helical content. The amino acid composition and N-terminal sequence of a protein independently prepared from bovine heart mitochondria, which is essential to the formation of the cytochrome c1-c complex, suggested that this colorless factor and the present heme-not-containing protein are identical. Evidence shows that another protein, called the non-heme protein, isolated from "two-band" cytochrome c1 preparation is also the same protein as that presented in this paper.