Reverse transcriptases contain a highly conserved YXDD amino acid motif believed to be important in enzyme function. The second amino acid is not strictly conserved, with a methionine, valine or alanine occupying the second position in reverse transcriptases from various retroviruses and retroelements. Recently, a 3.5-A (0.35-nm) resolution electron density map of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase positioned the YMDD motif within an antiparallel beta-hairpin structure which forms a portion of its catalytic site. To further explore the role of methionine of the conserved YMDD motif in HIV-1 reverse transcriptase function, we have substituted methionine with a valine, alanine, serine, glycine, or proline, reflecting in some cases sequence motifs of other related reverse transcriptases. Wild-type and mutant enzymes were expressed in Escherichia coli, partially purified by phosphocellulose chromatography, and assayed for the capacity to polymerize TTP by using a homopolymeric template [poly(rA)] with either a DNA [oligo(dT)] or an RNA [oligo(U)] primer. With a poly(rA).oligo(dT) template-primer, reverse transcriptases with the methionine replaced by valine (YVDD), serine (YSDD), or alanine (YADD) were 70 to 100% as active as the wild type, while those with the glycine substitution (YGDD) were approximately 5 to 10% as active. A proline substitution (YPDD) completely inactivated the enzyme. With a poly(rA).oligo(U) template-primer, only the activity of mutants with YVDD was similar to that of the wild type, while mutants with YADD and YSDD were approximately 5 to 10% as active as the wild-type enzyme. The reverse transcriptases with the YGDD and YPDD mutations demonstrated no activity above background. Proviruses containing the reverse transcriptase with the valine mutation (YVDD) produced viruses with infectivities similar to that of the wild type, as determined by measurement of p24 antigen in culture supernatants and visual inspection of syncytium formation. In contrast, proviruses with reverse transcriptases containing the YADD and YSDD mutations were less infectious than wild-type virus. These results point to the critical role of methionine of the YMDD motif in the activity of HIV-1 reverse transcriptase and subsequent replication potential of the virus.