Nucleotide segment (+169)AAAA(+172) constitutes an A-rich loop within human immunodeficiency virus type 1 (HIV-1) (HXB2D) RNA and is able to interact with the anticodon loop (33)/USUU(36) of primer tRNA3(Lys). We have shown that the deletion of this A-rich loop resulted in diminished levels of infectivity and reduced synthesis of viral DNA in MT-2 cells and cord blood mononuclear cells. Endogenous reverse transcriptase (RT) assays revealed that the mutated viruses, termed HIV/del-A, generated fewer cDNA products than did wild-type virus, designated HIV/WT. We also employed in vitro RT assays with in vitro-synthesized viral RNA templates, recombinant HIV-1 RT(p66/51), and natural tRNA3(Lys) as primers to show that the mutated RNA templates, designated PBS/del-A, generated less minus-strand strong-stop DNA product than did the wild-type RNA template, designated PBS/WT. The initiation efficiency of reverse transcription from the mutated RNA template was significantly impaired compared with that from the wild-type RNA template when a single-base extension assay from the tRNA3(Lys) primer was employed. However, RT reactions performed with DNA oligonucleotides complementary to the primer binding site (PBS) as primers did not yield differences between the mutated PBS/del-A and wild-type RNA templates. Long-term culture of HIV/del-A in MT-2 cells resulted in the replacement of two G's at nucleotide positions 167 and 168 by two A's that possessed the same relationship to the 5' end of the PBS as did the wild-type A's at positions 171 and 172. In vitro RT assays performed with recombinant enzyme with tRNA3(Lys) as the primer showed that the RNA template thus generated, termed PBS/A2, yielded levels of minus-strand strong-stop DNA product similar to those yielded by the wild-type RNA template. Coincidentally, viruses containing A's at positions 167 and 168 were able to replicate with efficiencies similar to those of the wild-type viruses. Thus, the (+169)AAAA(+172) A-rich loop plays a key role in the synthesis of viral DNA.