Clinical reports indicate that the development of drug resistance to AZT after chronic administration is common. In order to study this phenomenon, the T-cell line Jurkat E6–1 was treated continuously in vitro with low, gradually increased, concentrations of azidothymidine (AZT). Initially, 1 μM AZT significantly retarded the cell line from reaching confluence. However, after 10 weeks the T-cell line was able to grow in 10 μM AZT without any evidence of growth inhibition. Subsequently, cell isolates could grow continuously in the presence of 20, 50, and 100 μM AZT without growth inhibition. These T-cell lines (Jurkat E6–1/AZT-10, Jurkat E6–1/AZT-20, Jurkat E6–1/AZT-50, and Jurkat E6–1/AZT-100) were tested for AZT anabolism using purified [3H]AZT, and the results were compared to the wild-type untreated Jurkat E6–1 cell line. Similar intracellular AZT anabolites concentrations were determined in all cell lines. However, a four- to sixfold lower cellular concentration of mono-, di-, and triphosphate anabolites of AZT was determined in the Jurkat E6–1/AZT-10 cell line after 1 μM incubation and 6.5-fold lower after 10 μM AZT treatment. In general, a five- to sixfold reduction in the phosphorylation rates were estimated in the AZT resistant T-cell line. Pharmacology studies of [3H]AZT in the Jurkat E6–1/AZT-100 cell line showed a much lower level of activation of the pro-drug (28-fold), due to lack of thymidine kinase (TK) activity when compared to the Jurkat E6–1/AZT-10 T-cell line. A similar level of resistance was obtained at the thymidylate (dTMP) kinase level. Concurrently an additional mode of resistance (407-fold) was seen on the incorporation of the AZT triphosphate anabolite (AZTTP) into cellular DNA. The formation of this cell line in a period of ≤4 months coincides with the evidence of the clinical development of “resistance” to AZT in patients who receive the drug continuously. In addition, these T-cell lines have been infected with HIV, and studies on the development of collaterally sensitive regimens are under way.