Rat hepatic stellate cells (HSC) cultured in serum-containing medium underwent a rapid (3-hour) classical induction of p50:p65 and p65:p65 nuclear factor-κB (NF-κB) dimers. Subsequent culturing was associated with prolonged expression of active p50:p65 and persistent induction of a high-mobility NF-κB DNA binding complex consisting of potentially novel Rel-like protein(s). Formation of the latter complex was competed for by specific double-stranded oligonucleotides, was up-regulated by treatment of HSCs with tumor necrosis factor α (TNF-α), and was maintained at basal levels of expression by a soluble HSC-derived factor. An NF-κB–responsive CAT reporter gene was highly active in early cultured HSCs but was also trans-activated at a lower but significant level in longer-term cultured cells and could be completely suppressed by expression of dominant negative IκB-α. Physiological significance of the lower persistent NF-κB activities was also demonstrated by the ability of long-term cultured HSCs to support the activity of the NF-κB–dependent human intercellular adhesion molecule-1 (ICAM-1) promoter. Freshly isolated HSCs expressed high levels of IκB-α and IκB-β. Culture activation was accompanied by a long-term reduction in levels of IκB-α with no detectable expression in the nuclear fraction of cells, under these conditions p50:p65 was detected in the nucleus. IκB-β expression was transiently reduced and, upon replenishment, was associated with appearance of a lower-mobility IκB-β antibody-reactive species. Bcl3 expression was absent in freshly isolated HSC but was induced during culturing and became a persistent feature of the activated HSC. Inhibition of NF-κB DNA binding activity by gliotoxin was associated with increased numbers of apoptotic cells. We suggest that activation of NF-κB in cultured HSC is required for expression of specific genes associated with the activated phenotype such as ICAM-1 and may be antiapoptotic for rat HSCs.