Both clinical and animal studies have shown that angiogenesis is impaired in diabetes mellitus; however, the mechanisms responsible for this effect are poorly characterized. The major aims of the present study were to evaluate the effect of hyperglycemia on fibroblast growth factor 2 (FGF2)-induced angiogenesis in vivo and to determine whether FGF2 non-enzymatic glycation occurs in hyperglycemic mice. New blood vessel formation was examined in reconstituted basement membrane protein (Matrigel) plugs containing FGF2 in control normoglycemic CD1 and in hyperglycemic nonobese diabetic (NOD) mice. FGF2-induced angiogenesis in NOD mice was inhibited by 75% versus control mice (P < 0.001). When recombinant FGF2 was mixed with Matrigel and injected in mice, it was found that recombinant FGF2 glycation was significantly enhanced in plugs from NOD versus control mice (P < 0.01). In the Boyden chamber assay, the chemotactic effect of glycated FGF2 toward endothelial cells was lower than that of unmodified FGF2 (P < 0.01). Further, FGF2 glycated in vitro and co-injected with Matrigel in CD1 mice was a weaker angiogenic stimulus than unglycated FGF2 (P < 0.005). These results indicate that FGF2-induced angiogenesis is inhibited in diabetic mice, FGF2 glycation is enhanced in hyperglycemic mice, and glycation markedly reduces FGF2 chemotactic effect in vitro and its angiogenic properties in vivo. Thus, FGF2 glycation may represent a mechanism responsible for the impairment of angiogenesis in diabetes mellitus.