3'-Azido-3'-deoxythymidine (AZT) was the first anti-retroviral nucleoside analog to be used in the treatment and prevention of acquired immunodeficiency syndrome (AIDS). A novel (32)P-postlabeling assay, based upon thymidine kinase (TK) instead of the conventional T(4) polynucleotide kinase, has been developed for the detection of the levels of AZT incorporated into DNA. After enzymatic digestion of DNA to deoxynucleoside 3'-monophosphates, AZT was isolated by ethyl acetate extraction. The ethyl acetate was evaporated and the AZT was postlabeled by 5'-phosphorylation with [gamma-(32)P]ATP and TK. AZT was detected at a level of 51.5+/-6.3 (mean+/-SD; n=4) AZT molecules/10(5) nucleotides following in vitro incorporation of the drug into high-molecular-weight rat-liver DNA. The (32)P-postlabeling method was further validated by the detection of AZT in lung and liver DNA from neonatal mice treated with AZT. The levels of AZT in lung and liver DNA were proportional to the dose, with the levels in lung DNA being two-fold higher than those for liver DNA. The limit of detection for the assay was 8 AZT molecules/10(7) nucleotides using 10 microg of DNA.