AbstractNucleoside analogs were first approved by the U.S. Food and Drug Administration for use against HIV‐AIDS in 1987. Since then, these agents, now commonly referred to as nucleoside reverse transcriptase inhibitors (NRTIs), have become essential components of the Highly Active Antiretroviral Therapy (HAART) drug combinations used for treatment of Human Immunodeficiency Virus‐1 (HIV‐1) infections. Their antiretroviral activity is likely two‐fold: incorporation of the drug into viral DNA and inhibition of the viral reverse transcriptase. However, incorporation of the drug into host nuclear and mitochondrial DNA may be largely responsible for dose‐limiting toxicities. Azidothymidine (AZT, 3′‐azido‐3′‐deoxythymidine, zidovudine), the first NRTI approved for the therapy of HIV‐1, is incorporated into DNA, causes mutations in the hypoxanthine‐guanine phosphoribosyl‐transferase (HPRT) and thymidine kinase (TK) genes, and induces micronuclei, chromosomal aberrations, sister chromatid exchange, shortened telomeres, and other genotoxic effects in cultured cells. Genomic instability would be predicted as a consequence of these events. Metabolic pathways that result in the phosphorylation of AZT play a crucial role in AZT‐DNA incorporation, and may be altered after prolonged treatment. For example, thymidine kinase 1, the enzyme responsible for AZT mono‐phosphorylation, is down‐regulated during long‐term exposure and appears to be associated with AZT‐induced replication inhibition and the accumulation of cells in S‐phase. Detailed information on the mechanisms underlying NRTI‐associated antiretroviral efficacy, toxicity, and metabolic resistance were not available when AZT was first approved for use as an antiretroviral agent. Current insights, based on 15 years of research, may lead to intervention strategies to attenuate toxicity without altering drug efficacy. Environ. Mol. Mutagen., 2006. Published 2006 Wiley‐Liss, Inc.