Guanylate-binding proteins (GBPs) were originally described as proteins that are strongly induced by interferons and are capable of binding to agarose-immobilized guanine nucleotides, hGBP1, the first of two members of this protein family in humans, was recently shown to represent a novel type of GTPase that hydrolyzes GTP predominantly to GMP. We now report that purified recombinant hGBP2 also hydrolyzes GTP very efficiently, although GDP rather than GMP was the major reaction product. The biochemical parameters of this reaction were as follows: Krn = 313 lEVI, turnover number = 22 min -1. Both hGBP1 and hGBP2 failed to hydrolyze GDP, however, GDP was an effective inhibitor of the hGBP2-but not the hGBPl-catalyzed GTP hydrolysis reaction. Thus, hGBP1 and hGBP2 have similar biochemical properties, but show pronounced differences in product specificity.