6-Phosphogluconate dehydrogenase band II isoenzyme was isolated from a tobacco callus culture line, Nicotiana tabacum L., var. Virginia 115, utilizing ammonium sulfate precipitation, DEAF-cellulose chromatography, Red Sepharose CL-6B affinity chromatography followed by Sephadex G-150 gei filtration. N-terminal amino acid of the purified enzyme by dansylation method was lysine. Amino acid analysis indicated that the enzyme had 11 lysine and 17 arginine per 45,000 molecular weight subunit. Approximately 30 ninhydrin-positive spots were observed from tryptic peptide mapping study. Therefore, the results of N-terminal amino acid analysis, amino acid composition, and tryptic petide mapping suggest that the dimeric 6PG DH is composed of chemically identical subunits.