Human immunodeficiency virus (HIV) resistance to the nonnucleoside reverse transcriptase inhibitors emerges very rapidly under selection in culture and in patients. In contrast, zidovudine (3'-azido-3'-deoxythymidine [AZT])-resistant HIV generally emerges in patients only after more-prolonged therapy. Although HIV can be cultured from many patients shortly after the initiation of AZT treatment, characterization of the virus that is cultured generally indicates that it is sensitive to AZT. To initiate an evaluation of the mechanisms contributing to early HIV breakthrough in the presence of AZT and other nucleoside analogs, we have utilized replication-defective HIV encoding reporter genes. These recombinant HIV allow a quantitative analysis of a single cycle of infection. Results with these defective HIV indicate that early infection in the presence of AZT often results from the infection of a cell which is refractory to the antiretroviral effects of AZT. Characterization of a cell line derived from one such cell has demonstrated decreased accumulation of AZT triphosphate, increased phosphorylation of thymidine to thymidine triphosphate, and increased levels of thymidine kinase activity. In addition, AZT inhibition of replication-competent HIV infection is also significantly impaired in this cell line. Attempts to detect and characterize the mechanisms responsible for early viral infection after initiation of AZT therapy may result in the development of new strategies for prolonged suppression of viral infection prior to the emergence of drug-resistant virus.