The HIV-1 LTR promoter proximal G/C box array has been demonstrated to function by interacting with the Spl transcription factor family whose members can act as either activators or repressors of transcription. In this regard, we have examined the interaction of the HIV-1 Sp binding sites with nuclear factors that are present in cell types that support HIV-1 replication, including those of lymphocytic, monocytic, and astrocytic origin. As determined by electro-phoretic mobility shift (EMS) competition analyses using oligonucleotides containing the sequences of each of the Spl sites of HIV-1 strain LAI, the NF-kB-proximal Sp site (site III) displayed the highest binding activity compared to sites I and II with regard to Spl and related factors present in lymphocytic (Jurkat) and astrocytic (U-373 MG) nuclear extracts. Spl and two Sp3 isoforms were detected as the primary cellular constituents of DNA-protein complexes formed with the NF-JcB-proximal site. Only modest differences in Spl:Sp3 binding ratios were observed when this site was reacted with either astrocytic or lymphocytic nuclear extract. However, when nuclear extracts derived from two monocytic cell types that differ in the degree of differentiation were reacted with the HIV-1 LAI Sp site III, a large difference in Spl and Sp3 binding was observed. To determine if naturally occurring and replication-competent strains of HIV-1 contain base pair alterations within the Sp elements that affect the ability of the site to interact with Spl and related factors, a series of Sp site III variants were constructed and examined by EMS analyses. One of these sites, obtained from the published sequence of the YU-2 strain (a brain-derived macrophage tropic strain of HIV-1), displayed almost no Spl or Sp3 binding activity as a result of a single base pair alteration in Sp site III. This base-pair alteration, when placed in the context of an HIV-1 LAI LTR-luciferase construct, resulted in a 40-50% reduction in LTR activity in transiently transfected Jurkat and U-373 MG cells. Overall, these results suggest that specific G/C box sequence alterations present in the brain-derived HIV-1 variant YU-2, or possibly other brain-derived variants, may exhibit altered replication properties as a result of the low affinity of the NF-KB-proximal G/C box for members of the Sp transcription factor family