HIV-1 viral production is restricted intracellularly in astrocytes compared with lymphocytes due to the limited expression of viral structural proteins. The poor translation of HIV-1 mRNA and consequent limited virion production can be restored by overexpression of TRBP proteins in the astrocytoma U251MG cells. TRBP1 and TRBP2 are double-stranded RNA binding proteins that increase HIV-1 gene expression. Both proteins are produced from a single gene that possesses two independent promoters and an alternative first exon. Endogenous expression is restricted in astrocytes due to limited TRBP promoter expression compared to lymphocytes. We examined the transcriptional regulation of TRBP1 and TRBP2 by in vivo genomic footprinting in the lymphocytic Jurkat and in the astrocytic U251MG cells. We identified one AP4 and one AP2-binding site that regulate the TRBP2 promoter in both cell types, and one Sp1 and two CCAAT-binding sites that control TRBP1 expression. Mutations in the TRBP1 promoter modulate its expression specifically in Jurkat and in U251MG. The analysis of the CCAAT-390 site by EMSA and by ChIP demonstrates that NF-Y/CBF transcription factor binds specifically to the promoter in vitro and in vivo. Furthermore, each NF-Y subunit was more highly expressed in the lymphocytic cells, compared to astrocytic cells. An NF-YA trans-dominant mutant decreased TRBP1 promoter expression fourfold in Jurkat cells, thus demonstrating the functional importance of NF-Y factors in lymphocytes. These studies suggest that the cell specifity of HIV-1 expression and replication may be regulated, in part, through the control of TRBP1 expression.