Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution. Acidic fibroblast growth factor (aFGF)1 is considered to be involved in several important physiological and pathological processes, such as angiogenesis, wound healing (1), and atheromatosis (2-6). In cell cultures it can stimulate growth, cell migration, and differentiation. As the growth factor lacks a classical signal sequence, its mechanism of secretion is still unclear, but it appears to involve a pathway different from the classical secretion pathway through the endoplasmic reticulum and Golgi apparatus (7-9). At the cell surface, aFGF binds with high affinity to transmembrane FGF-receptors (FGFR) containing a cytoplasmic split tyrosine kinase domain. There are four different genes encoding FGFR (FGFR1-4) with several different splicing variants. aFGF also binds to heparan sulfate proteoglycans at the cell surface, although with lower affinity. Upon activation, the FGFR is phosphorylated on tyrosine residues, and it activates downstream effectors such as phosholipase C␥ and the mitogen-activated protein kinase pathway. There are some conflicting data as to whether phosphatidylinositol (PI) 3-kinase is also activated (10 -13). After binding to high affinity cell surface receptors, aFGF is translocated across cellular membranes and transported to the nucleus (14 -22). The mechanism of this process is not well defined. It depends on FGFR and, at least in the case of FGFR4, also on the intactness of the C terminus of its cytosolic part. 2Human PI 3-kinases phosphorylate PI in the 3Ј position of the inositol ring and can be divided into three main classes on the basis of in vitro substrate specificity (23). Class I phosphorylates PI, PI 4-phosphate and PI 4,5-diphosphate and include...