Guanosine 5'-triphosphate (GTP) plays a significant role in the bioenergetics, metabolism, and signaling of cells; consequently, any modifications to the structure of the molecule can have profound effects on a cell's survival and function. Previous studies in our laboratory demonstrated that like proteins, purines, and pyrimidines can nonenzymatically react with sugars to generate advanced glycation endproducts (AGEs) and that these AGEs can form in vitro under physiological conditions. The objective of this investigation was twofold. First, it was to evaluate the susceptibility of ATP, GTP, CTP, and TTP to nonenzymatic modification by D-glucose and DL-glyceraldehyde, and second to assess the effect of various factors such as temperature, pH and incubation time, and sugar concentration on the rate and extent of nucleotide triphosphate AGE formation. Of the four nucleotide triphosphates that were studied, only GTP was significantly reactive forming a heterogeneous group of compounds with DL-glyceraldehyde. D-Glucose exhibited no significant reactivity with any of the nucleotide triphosphates, a finding that was supported by UV and fluorescence spectroscopy. Capillary electrophoresis, high-performance liquid chromatography and mass spectrometry allowed for a thorough analysis of the glycated GTP products and demonstrated that the modification of GTP by dl-glyceraldehyde occurred via the classical Amadori pathway.