A relatively conserved threonine residue corresponding to threonine 11 of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase is proposed to be involved in the interaction with the phosphate group of NAD+. Site-directed mutagenesis was used to examine the important role of this residue. Threonine 11 was changed ot alanine (T11A), cysteine (T11C), serine (T11S) or tyrosine (T11Y) and the mutant proteins were expressed in E. coli. Mutants T11A, T11C and T11Y were found to be inactive. Mutant T11S still retained substantial activity and the Km value for prostaglandin E2 was similar to the wild type enzyme;however, the Km value for NAD+ was increased over 23 fold. These results suggest that threonine 11 is critical for interaction with NAD+ and contributes to the full catalytic activity of 15-PGDH.