Abstract 5′-Nucleotides could be conveniently prepared in high yields by the phosphorylation of 2′, 3′-O-isopropylidene nucleosides with phosphoryl chloride in the absence of any organic solvent. The rate of phosphorylation in a large excess of phosphoryl chloride was markedly promoted by the partial hydrolysis of phosphoryl chloride with a small amount of water. The undesirable cleavage of the glycosidic linkage in nucleoside, which was observed particularly in a strongly acidic medium caused by the addition of excessive water, could be suppressed by the addition of ethers or amines in a phosphoryl chloride solution. 2′, 3′-O-Isopropylidene derivatives of adenosine, inosine, and guanosine gave the corresponding 5′-nucleotides in good yields, whereas that of xanthosine reacted very slowly, unlike the above nucleosides. All attempts at the phosphorylation of 2′, 3′-O-isopropylidene-2-mercaptoinosine were unsuccessful. The influence of substituents at the 2-position in purine bases on phosphorylation was investigated by comparing the reactivity between the above-mentioned nucleosides and their methylated derivatives, such as 2-dimethylaminoinosine, 2-methoxyinosine, and 2-methylthioinosine; it was found that these methylated nucleosides were readily phosphorylated to give the corresponding 5′-nucleotides in nearly quantitative yields.