Gel retardation assays with nuclear extracts from various cells and a double-stranded oligonucleotide representing the sequence of the HIV-1 enhancer revealed at least two proteins of 43 and 53K that interacted specifically with the HIV enhancer and differed from those reported before. A simple and fast procedure for purification of these proteins involving heparin-agarose and DNA affinity chromatography steps was developed. The electrophoretic data suggest that the proteins are sufficiently pure for sequencing once the procedure is scaled up.