A rapid, simple, and sensitive radiochemical assay for the measurement of purine or pyrimidine nucleoside kinases (EC 2.7.1.-) is described. The substrate (thymidine, deoxyuridine, deoxycytidine, deoxyguanosine, deoxyadenosine, uridine, cytidine, and adenosine) is separated from the product (the respective 5′-nucleotide) on neutral alumina columns which retain the nucleotides but not the nucleosides. The nucleotides are recovered by elution with 0.4 m sodium phosphate buffer, pH 7.6. © 1981.