Human immunodeficiency virus type 1 (HIV-1) isolates resistant to (−)-β-d-dioxolane-guanosine (DXG), a potent and selective nucleoside analog HIV-1 reverse transcriptase (RT) inhibitor, were selected by serial passage of HIV-1LAI in increasing drug concentrations (maximum concentration, 30 μM). Two independent selection experiments were performed. Viral isolates for which the DXG median effective concentrations (EC50s) increased 7.3- and 12.2-fold were isolated after 13 and 14 passages, respectively. Cloning and DNA sequencing of the RT region from the first resistant isolate identified a K65R mutation (AAA to AGA) in 10 of 10 clones. The role of this mutation in DXG resistance was confirmed by site-specific mutagenesis of HIV-1LAI. The K65R mutation also conferred greater than threefold cross-resistance to 2′,3′-dideoxycytidine, 2′,3′-dideoxyinosine, 2′,3′-dideoxy-3′-thiacytidine, 9-(2-phosphonylmethoxyethyl)adenine, 2-amino-6-chloropurine dioxolane, dioxolanyl-5-fluorocytosine, and diaminopurine dioxolane but had only marginal effects on 3′-azido-3′-deoxthymidine (AZT) susceptibility. However, when introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q), the K65R mutation reversed the AZT resistance. DNA sequencing of RT clones derived from the second resistant isolate identified the L74V mutation, previously reported to cause ddI resistance. The L74V mutation also decreased the AZT resistance when the mutation was introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q) but to a lesser degree than the K65R mutation did. These findings indicate that DXG and certain 2′,3′-dideoxy compounds (e.g., ddI) can select for the same resistance mutations and thus may not be optimal for use in combination. However, the combination of AZT with DXG or its orally bioavailable prodrug (−)-β-d-2,6-diaminopurine-dioxolane should be explored because of the suppressive effects of the K65R and L74V mutations on AZT resistance.