Cell surface heparan sulfate proteoglycans (HSPGs) mediate internalization of HIV-1 Tat. Herein, we report that human WiDr cells, which express perlecan but no other HSPGs, can internalize ¹²⁵I-labeled Tat with minimal lysosomal degradation. Pre-treatment of cells with heparitinase almost completely abolished ¹²⁵I-Tat surface binding, while the use of an HIV-1 long terminal repeat (LTR) promoter–reporter construct demonstrated that transactivation was potently blocked by pretreatment of cells with heparitinase, indicating an essential role for perlecan in the biologic effects of Tat. We conclude that the perlecan mediates Tat uptake and is required for HIV-1 LTR-directed transactivation in this human cell type.