The carbocyclic nucleoside 1592U89 is a selective inhibitor of the human immunodeficiency virus (HIV), targeting the reverse transcriptase (RT). In vitro selection studies were undertaken to generate resistant variants with both HIV type 1 (HIV-1) wild-type strain HIV-1(HXB2) and 3'-azido-3'-deoxythymidine (AZT)-resistant strain HIV-1(RTMC). At least two or three mutations in RT were required to produce a 10-fold reduction in susceptibility. The first RT mutation selected was at codon 184, methionine (M) to valine (V), for HIV-1(HXB2) and HIV-1(RTMC), conferring two- and fivefold resistance, respectively. Two additional mutations were selected with HIV-1(HXB2), either leucine (L) 74 to V and lysine (K) 65 to arginine (R) (first-passage series) or L74 to V and tyrosine (Y) 115 to phenylalanine (F) (second-passage series). Cloned variants, obtained from the 1592U89 selection, were either double RT mutants 65R/184V and 74V/184V or triple RT mutant 74V/115Y/184V. Molecular clones were constructed with single, double, and triple combinations of these mutations for resistance analysis with different RT inhibitors. Each individual mutation conferred only low-level resistance (two- to fourfold) to 1592U89 in the HXB2 background. Double mutants containing the 184V mutation and triple mutants showed slightly greater levels of resistance to 1592U89 (7- to 11-fold). Some of the 1592U89-resistant variants were cross-resistant with 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and (-)-2'-deoxy-3'-thiacytidine, but none were resistant to 2',3'-didehydro-3'-deoxythymidine or AZT.