Two biotin-labeled nucleotide analogs, Bio-4-dUTP and Bio-12-SS-dUPT, were synthesized by a modification of the procedure described by Langer et al. (1981). Deoxyuridine 5'-triphosphate was first mercurated at the 5-C and subsequently reacted with allylamine to form 5-(3-amino)allyldeoxyuridine 5'-triphosphate (AA-dUTP). AA-dUTP was purified and reacted with either N-hydroxysuccinimide-activated biotin to form Bio-4-dUTP, or with N-hydroxysuccinimide-activated 2-(biotinamido)ethyl-1,3'-dithiopropionate to form Bio-12-SS-dUTP. Bio-12-SS-dUTP is a chemically cleavable biotinylated nucleotide analog containing a disulfide bond in the 12-atom linker arm joining biotin to the pyrimidine base. Both biotinylated nucleotide analogs were purified either by ion-exchange chromatography or by ion-pair reverse-phase HPLC. Bio-4-dUTP was identified by (i) its unique absorbance spectrum, (ii) its coelution with 3H-Bio-4-dUTP during reverse-phase HPLC, and (iii) its ability to bind to avidin agarose. As a functional assay for both the synthesis and purification of the biotinylated nucleotide analogs, each nucleotide was incorporated into DNA by nick-translation. The nick-translated DNA was shown to contain biotinylated nucleotides by its ability to bind to biotin-cellulose affinity columns following incubation with soluble avidin. DNA nick-translated in the presence of Bio-12-SS-dUTP was recovered from the biotin-cellulose column following incubation in buffer containing 50 mM dithiothreitol. The susceptibility of the disulfide bond in the linker arm of Bio-12-SS-dUTP to cleavage by dithiothreitol was shown to be unaffected by the presence of avidin bound to the biotin group.