Glyceraldehyde-3-phosphate dehydrogenase with a specific activity of 153 units/mg protein was isolated from bovine heart muscle. Its relative molecular mass was found to be 144,000. The tryptic peptide map and amino acid analysis were obtained. The N-terminal sequence was established as Val-Lys-Val-Gly-Val-Asn-Gly-... and C-terminal as ...-Ala-Ser-Lys-Glu. Fluorescence and optimal rotation dispersion measurements were performed. The data were compared with other glyceraldehyde-3-phosphate dehydrogenases.