Hypobromous acid (HOBr) is generated not only by eosinophils but also by neutrophils in the presence of Br⁻ at the plasma concentration. Reactivity of HOBr is greatly modulated by coexistent compounds such as amines and amides. In this study, we investigated effects of urea in the reaction of nucleosides with HOBr. When nucleosides were incubated with HOBr without urea in potassium phosphate buffer at pH 7.4 and 37°C, the reactions almost completed within 10 min, with consumptions in the order of 2′-deoxyguanosine > 2′-deoxycytidine > 2′-deoxythymidine > 2′-deoxyadenosine, generating 8-bromo-2′-deoxyguanosine and 5-bromo-2′-deoxycytidine. In the presence of urea, the reaction of nucleosides with HOBr was relatively slow, continuing over several hours. When HOBr was preincubated without urea in potassium phosphate buffer at pH 7.4 and 37°C for 48 h, the preincubated HOBr solution did not react with nucleosides. However, a similar preincubated solution of HOBr with urea reacted with nucleosides to generate 8-bromo-2′-deoxyguanosine and 5-bromo-2′-deoxycytidine. These results imply that a reactive bromine compound with a long life, probably bromourea, is generated by HOBr in neutral urea solution and reacts with nucleosides, resulting in brominated nucleosides.