A compensatory mutation (M230I) in the primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) restores the replication capacity of virus having a Y115W mutation in their RT coding region. The Y115W substitution impairs DNA polymerase activity and produces an enzyme with a lower fidelity of DNA synthesis. Gel-based fidelity assays with the double mutant Y115W/M230I revealed that the M230I substitution increased the accuracy of mutant Y115W. Y115W/M230I showed wild-type misinsertion fidelity in assays performed with DNA/DNA templates. However, when present alone, M230I conferred reduced fidelity as determined in misinsertion and mispair extension fidelity assays, as well as in primer extension assays carried out with three dNTPs. The mutant M230I showed a 3.3-16-fold increase in misinsertion efficiency for G, C and T opposite T, compared with the wild-type enzyme. Its fidelity was not influenced by nucleotide substitutions in the template/primer around the incorporation site. However, its accuracy was apparently affected by the structure of the 5'-overhang of the template strand. Unlike wild-type HIV-1 RT, nucleotide selectivity of mutant M230I at dT:dG, dT:dC and dT:dT mispairs was almost exclusively dependent on the K(m) values for correct and incorrect dNTPs, a characteristic that has not been described for other low fidelity mutants of HIV-1 RT.