Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with a variety of malignancies including nasopharyngeal carcinoma. The EBV-encoded latent membrane protein 1 (LMP1) is considered the EBV oncogene as it is necessary for EBV-induced B-lymphocyte transformation and has been shown to transform rodent fibroblasts. LMP1 contains two signaling domains, the carboxy-terminal activating region 1 and 2 (CTAR1 and CTAR2), by which NF-kappaB, phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase, and c-Jun N-terminal kinase are activated. In this study, the role of CTAR1 and CTAR2 in LMP1-mediated transformation of rodent fibroblasts was analysed. CTAR1 was found to be necessary for rodent fibroblast transformation, whereas CTAR2 was dispensable. The activation of the PI3K pathway in Rat-1 cells by LMP1 and LMP1-CTAR1 in transformed cells resulted in phosphorylated Akt and phosphorylated glycogen synthase kinase 3beta. The role of PI3K and NF-kappaB activation in LMP1-mediated transformation was further analysed using the chemical inhibitors LY294002 and BAY 11-7085. LY294002 inhibited CTAR1-induced focus formation and anchorage-independent growth, whereas BAY 11-7085 did not inhibit focus formation or anchorage-independent growth. Similar studies in human fibroblasts confirmed that LMP1-CTAR1 also mediates aberrant growth, phosphorylation of Akt, and decreased levels of p27. These findings indicate that LMP1-mediated rodent fibroblast transformation is dependent upon activation of PI3K and Akt and is independent of activation of NF-kappaB.