Hypoxanthine-guanine phosphoribosyltransferase (HGPT) occurs in both eukaryotic and prokaryotic organisms. However, the molecular and functional properties of plant HGPT are not well understood. In this study, it was found that the putative HGPT proteins from dicot and monocot plant species exhibited significant identities to their homologs from other cellular organisms. Ectopic expression of the HGPTs from Arabidopsis, soybean or wheat complemented HGPT deficiency in the hpt1 mutant of Saccharomyces cerevisiae. Recombinant Arabidopsis HGPT (AtHGPT) catalyzed both forward and reverse reactions in in vitro biochemical assays. The relative catalytic efficiency for the synthesis of guanosine monophosphate (GMP) was significantly greater than that for the production of guanine from GMP. Further investigations led to identification of the candidate residues that may form the pyrophosphate (PPi) binding loop in AtHGPT. AtHGPT expression level was dynamically regulated in Arabidopsis organs and during leaf development and senescence and seed germination. AtHGPT knockout mutant germinated more slowly than wild type control, whereas its overexpression mutant exhibited accelerated germination. Collectively, the data suggest that functional HGPTs are expressed in higher plants. In Arabidopsis, HGPT plays an active role in the salvage of purine bases and its activity is required for efficient seed germination.