The subcellular distribution of endogenous basic fibroblast growth factor (bFGF) was studied in the human hepatoma cell line, SK Hep-1. Basic FGF was demonstrated in cytosol, nuclei, and membranes by purification from each subcellular fraction using ion-exchange chromatography and heparin-affinity chromatography, and by the detection of bFGF-immunoreactive proteins on Western blots of heparin-affinity purified samples. About 65% of bFGF bioactivity was present in cytosol, 17% in nuclei, and 18% in membranes. Antisera raised against either recombinant 18 kDa bFGF or a bFGF N-terminal extension peptide showed that cytosol contained bFGF of mainly Mr 18,000 whereas nuclei and membranes contained three forms of bFGF of Mr 18,000, 22,500, and 24,000. Mitogenic activity in nuclei was chromatin-associated and required 0.6 M NaCl or 100 micrograms/ml heparin for maximal release. Membrane-bound activity was released by 0.6 M NaCl but not by heparin. The finding that endogenous bFGF proteins are present in various subcellular compartments suggests that bFGF may have additional biological roles at these sites.