Single-base deletions at nucleotide runs or –1 frameshifting by HIV-1 reverse transcriptase (RT) result from template slippage during polymerization. In crystal structures of HIV-1 RT complexed with DNA-DNA template-primer, the palm subdomain in the template cleft contacts template backbone near the proposed site of slippage via the Glu89 sidechain. We investigated the role of residue Glu89 in frameshifting by perturbing this interaction. Substitutions with Asp, Gly, Ala, Val, Ser, Thr, Asn, or Lys were created in recombinant HIV RT, and frameshift frequencies of resulting mutant RTs were measured. All substitutions led to reduced –1 frameshifting by HIV-1 RT (2- to 40-fold). Interestingly, the suppression of –1 frameshifting frequently coincided with an enhancement of +1 frameshifting (3- to 47-fold) suggesting that residue Glu89 can influence the slippage of both strands. Glu89 substitutions also led to reduced rates of dNTP misincorporation that paralleled reductions in -1 frameshifting, suggesting a common structural mechanism for both classes of RT error. Our results reveal a major influence of residue Glu89 on slippage-mediated errors and dNTP incorporation fidelity. Crystal structure of HIV-1 RT reveals a salt bridge between Glu89 and Lys154, which may facilitate –1 frameshifting; this concept is supported by the observed reduction in –1 frameshifting for Lys154A and Lys154R mutants.