Foscarnet (PFA) is a pyrophosphate analogue antiviral active against human immunodeficiency virus (HIV-1) and herpesviruses. Strains of HIV-1 resistant to PFA have mutations in the HIV-1 reverse transcriptase (RT). We examined the influence of PFA resistance mutations, in different genetic backgrounds, on HIV-1 replication competency in both replication kinetics and growth competition assays. In replication kinetics assays, the recombinant strains HX89K, HX92I, and HX156A (encoding RT mutations E89K, L92I, and S156A, respectively, in the HXB2-D genetic background) replicated to lower titers than the wild-type parent in the absence of drug, and the degree of replication impairment increased as PFA resistance increased. PFA-resistant strains LAI 92I and LAI 156A (encoding RT mutations L92I and S156A, respectively) were replication impaired in comparison to the wild-type parent LAI to a similar degree as observed for strains in the HXB2D background. In growth competition assays with wild-type LAI, strains LAI 92I and LAI 156A had relative fitness values of 0.5 and 0.8, respectively. These results show that the RT mutations E89K, L92I and S156A, observed in PFA-resistant strains selected in cell culture, reduce replication competence. Furthermore, these data show a correlation of increasing PFA resistance and decreasing replication competence mediated by single amino acid substitutions in the RT.