The human polyomavirus, JCV, differs from other papovaviruses in its tissue tropism for human glial cells. Transcription of the early region of the virus, at least in part, contributes to the tissue specificity of JCV. In this study, we have synthesized oligonucleotides which span the JCV 98 bp repeat unit. Using gel mobility shift and UV cross-linking assays, we have demonstrated that four proteins from a human fetal brain extract interact specifically with the JCV promoter/enhancer. Two proteins of 82 kd and 78/80 kd recognize the 5'- and 3'-terminal regions of the JCV 98 bp repeat sequence, respectively. The mol. wt of these proteins are similar in HeLa and brain extracts. In contrast, the proteins which recognize the central region of the 98 bp enhancer are distinct in HeLa (85 kd) and fetal brain (45 kd) extracts. The possible role of these proteins in tissue-specific expression of the JCV early promoter in brain cells is discussed.