The human neurotropic virus JCV contains a 98-base pair repeat enhancer/promoter sequence that confers glial-specific transcription to the viral early and late promoters. The central region of this repeat, designated the B-domain, binds to a glial-derived nuclear protein that stimulates transcription of the viral promotor in vitro. We now report the isolation of a recombinant cDNA clone, termed glial factor-1 (GF1), from a brain expression library that encodes a novel protein which interacts with the JCV B-domain. Results from RNA studies indicate that the GF1 transcript is more abundant in brain than in other tissues and that the level of GF1 RNAs increases progressively during brain development. Cotransfection of the recombinant GF1 expressor plasmid with JCV promoters indicates that GF1 stimulates transcription of the JCV late promoter and to a lesser extent the JCV early promoter predominantly in cells of human glial origin. Thus, GF1 is a sequence-specific DNA binding protein that may play a role in determining the glial-specific expression of JCV.