7-Deazaxanthine (7-DX) was recently identified as the first purine derivative with pronounced inhibitory activity against Escherichia coli thymidine phosphorylase (TP) and angiogenesis. In order to "freeze" the enzyme in an open, inactive conformation, a novel multi-substrate analogue inhibitor of TP, containing an alkyl phosphonate moiety covalently linked to 7-DX, was synthesized. The prototype compound TP65 (9-(8-phosphonooctyl)-7-deazaxanthine) (at 250 microM) completely inhibited TP-induced formation of microvascular sprouts from endothelial cell aggregates in a three-dimensional fibrin gel. In the chick chorioallantoic membrane assay, TP caused a dose-dependent stimulation of angiogenesis, which was completely inhibited by 250 nmol TP65. This dose proved to be non-toxic for the developing chick embryo. TP65 thus emerges as a potent and specific inhibitor of TP and TP-induced angiogenesis, which opens new perspectives for multi-substrate analogue inhibitors of TP as potential anti-cancer agents and as inhibitors of angiogenesis and of diseases with enhanced expression of TP.