We have analyzed protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1 in a productively infected T-cell line by in vivo dimethyl sulfate footprinting. Major footprints are evident at the basal promoter and enhancer elements. In particular, proteins appear to occupy the TATA box, the Sp1 sites, and the two repeats of the enhancer region. In the negative regulatory element, protections are detected over the USF/MLTF and NFAT-1 sites. Furthermore, two previously unrecognized sites, from nucleotides -260 to -275 and from nucleotides -205 to -216, respectively, appear to be involved in protein-DNA interactions. These two sites are purine rich and share a common sequence motif.