Several pyrimidine nucleosides and polydeoxyribonucleotides have been examined with respect to their ultraviolet absorption spectra and the kinetics of their decomposition under the conditions used in the C- and (C + T)-specific treatments of the sequencing procedure of Maxam and Gilbert. In hydrazine-water (56:44, v/v), at 20 degrees C, thymidine was found to be virtually fully ionized. Under these conditions, the susceptibility to hydrazinolysis decreased in the order N3-methylthymidine greater than uridine approximately equal to deoxyuridine greater than deoxycytidine approximately equal to polydeoxycytidylate greater than polythymidylate much greater than thymidine greater than 5-methyldeoxycytidine. Addition of sodium chloride to 1.5 M slightly accelerated hydrazinolysis of those nucleosides bearing nonionizable heterocyclic moieties and retarded hydrazinolysis severalfold for those nucleosides containing an ionizable aglycone (thymidine, deoxyuridine, uridine). It is concluded that the nucleosides of this latter class react with hydrazine largely or exclusively in their nonionized form and that the main effect of added salt is increased ionization of the heterocycles, resulting in a decreased population of nonionized nucleosides.