We have shown that the EBV oncoprotein, latent membrane protein 1 (LMP1), induces a constellation of tumor-invasiveness factors. Fibroblast growth factor (FGF)-2 is angiogenic as well mitogenic. Although FGF-2 does not contain a hydrophobic signal sequence for secretion, FGF-2 is released extracellularly. However, the mechanism by which FGF-2 is released is unclear. Here we show first that LMP1 induces in epithelial cells the expression of FGF-2 mRNA and protein through both LMP1 COOH-terminal activation domains, CTAR 1 and CTAR 2, which can activate nuclear factor (NF)-kappaB signaling and also the p38 mitogen-activated protein kinase pathway. Coexpression of IkappaBalpha (S32A/S36A), which cannot be phosphorylated and prevents NF-kappaB activation, with LMP1 inhibited induction of FGF-2 by LMP1, which suggests that LMP1 induces FGF-2 via NF-kappaB signaling. Moreover, unlike phorbol 12-myristate 13-acetate LMP1 also induced the release of the M(r) 18,000 isoform of FGF-2 protein. Transfection of Ad-AH cells with LMP1 deletion mutants lacking either CTAR 1 or CTAR 2 also induced the release of the protein. Secretion was confirmed in 293 cells, which do not contain detectable endogenous FGF-2 protein, cotransfected with FGF-2 and LMP1. Finally, Na(+)/K(+)-ATPase participates in FGF-2 release, independently of the classical endoplasmic reticulum/Golgi pathway. In this study, the release of M(r) 18,000 FGF-2 protein was partially suppressed by ouabain, which inhibits the activity of Na(+)/K(+)-ATPase alpha1 subunit, but not by Brefeldin A, which inhibits the endoplasmic reticulum/Golgi-dependent secretory pathway. In contrast, the release of M(r) 18,000 FGF-2 protein was almost completely inhibited by IkappaBalpha (S32A/S36A). These results suggest that FGF-2 release is independently mediated by NF-kappaB signaling, not simply a consequence of induction itself. Thus, NF-kappaB signaling is involved in induction of expression and release of FGF-2 by LMP1.