We have previously shown that multidrug-resistant cells expressing the multidrug transporter P-glycoprotein are less sensitive to the antiviral activity of AZT. Subsequently, we addressed the question whether AZT itself is able to induce cellular resistance to the drug. Indeed, CEM cells propagated in the presence of increasing concentrations of AZT become resistant to the antigrowth and antiviral activity of AZT but do not express detectable level of P-glycoprotein. Sensitivity of these cells to other compounds, such as vinblastine, vincristine, ddI, and ddC remained unchanged, indicating that, in contrast to P-glycoprotein-positive cells, AZT-induced resistance is specific for AZT. Interestingly, in AZT-induced resistant cells the intracellular accumulation of AZT and exogenous deoxythymidine, as well as thymidine kinase activity, are significantly reduced when compared with the parental cell line. Our findings show that AZT itself may directly induce the expression of cellular mechanisms leading to the acquisition of specific cellular resistance that can affect its antiviral activity.