BACKGROUND In this study, a simple and sensitive fluorometric HPLC method was described for the determination of nitrite, an indicator of nitric oxide production in biological samples. METHODS Nitrite was reacted with 2,3-diaminonaphthalene (DAN) to form fluorescent 2,3-naphthotriazole (NAT). Because NAT has higher fluorescence intensity at alkaline conditions, a reverse phase HPLC method employing a polymer-based column was applied to separate NAT using an alkaline mobile phase system. RESULTS NAT was well separated from DAN and other fluorescent substances with increased detection sensitivity under alkaline conditions. A linear relationship (r=0.999) between the fluorescence intensity of NAT and the concentration of nitrite was observed in the range from 0 to 800 nM with the detection limit of about 25 nM. CONCLUSIONS The present HPLC method appears suitable for routine analysis of nitrite in crude biological samples, since the polymer-based column can withstand rigorous cleaning with either acid or base.