Nucleoside reverse-transcriptase (RT) inhibitors (NRTIs), including lamivudine (3TC) and zidovudine (Zdv), are being evaluated for the treatment of human T cell lymphotropic virus type 1 (HTLV-1)-associated disease. However, information on the susceptibility of HTLV-1 to these drugs is limited. The activity of 5 NRTIs on HTLV-1 RT was evaluated. IC(50) values for Zdv, zalcitabine (ddC), didanosine (ddI), 3TC, and stavudine (d4T) were determined, using an enzymatic assay, for 5 HTLV-1 isolates and for reference wild-type and NRTI-resistant human immunodeficiency virus type 1 (HIV-1). Both HTLV-1 and wild-type HIV-1 were equally susceptible to Zdv, ddC, ddI, and d4T. In contrast, high-level resistance to 3TC was found in all HTLV-1 isolates. The findings support the clinical use of Zdv, ddC, ddI, and d4T but not of 3TC for the antiretroviral treatment of HTLV-1-associated disease.