Pentosidine is an advanced glycation end product, formed by oxidation and glycation that accumulates markedly during end-stage renal failure. Measurement of the pentosidine level in physiological samples is applied as a sensitive marker for the early diagnosis of renal failure. In the quantitative measurements of pentosidine reported to date, a rapid enzyme-linked immunosorbent assay (ELISA) has been widely used to estimate the plasma/serum pentosidine levels in a number of clinical samples, because high performance liquid chromatography (HPLC) methods require multiple preparation steps before the analysis. However, the currently used clinical analysis of the plasma/serum pentosidine level by ELISA requires incubation of the plasma/serum at 100°C for 15 min to inactivate the protease, which is required before the anti-pentosidine antibody can bind to the pentosidine. In the present study, we examined whether pentosidine could be generated artificially through the heating of serum. The pentosidine content, measured by HPLC, in the serum increased by heating in a temperature- and time-dependent manner. The pentosidine content was increased 1.1- to 4.2-fold by the heating process compared to unheated samples, and the increased rate was not identical for each sample. After removing low-molecular weight (<10,000) serum components, the heat-induced pentosidine formation was decreased. Furthermore, the increase in pentosidine formation was significantly inhibited by acidic conditions more than by the addition of diethylene triamine pentaacetic acid, a metal chelator. This indicates that the level of serum pentosidine will be measured more accurately by ELISA if hydrochloric acid is added during the heating process.