The signal transduction cascade initiated by the activation of phosphoinositide 3-kinase (PI-3 kinase) is implicated in mitogenic and antiapoptotic signaling generated by growth factors in a variety of cell types. We have examined the consequences of an inhibition of this pathway in human diploid fibroblasts. We find that a specific PI-3 kinase inhibitor (LY294002) causes growth arrest in these cells accompanied by changes in gene expression that are similar to those seen during cellular senescence. A second inhibitor, PD58029, which is specific for the mitogen-activated protein kinase kinase 1 (MEK-1), also induces a growth arrest but does not induce the same spectrum of gene expression. The pattern of gene expression in the presence the MEK-1 inhibitor is similar to that seen during growth arrest induced by serum starvation. The specific phenotypic changes seen following inhibition of PI-3 kinase are: an increase in beta-galactosidase activity; a decrease in EPC-1 gene expression; and a dramatic increase in collagenase gene expression. Thus, growth arrest with a PI-3 kinase inhibitor induces a senescent-like phenotype that is not seen when cells are growth arrested by either serum starvation or a MEK-1 inhibitor.