Unlike immortalized cell lines, normal human fibroblasts in culture undergo replicative senescence in which the number of population doublings is limited. While fibroblasts display a variety of changes as they senesce in vitro, little is known about how gene expression varies as a function of population doubling level. We have used differential hybridization screening to identify human genes that are preferentially expressed in senescent cells. While we found several isolates that were up-regulated in late-passage cells, all appeared to be variants of the same cDNA, which we named senescence-associated gene (SAG). Our data show that SAG expression is threefold higher in senescent fibroblasts and closely parallels the progressive slowdown in growth potential, but is not cell-cycle regulated. Thus, SAG serves as an accurate marker for fibroblast growth potential during replicative senescence. Further studies demonstrated that SAG is a novel gene active in nearly all tissue types tested and that it is conserved through evolution. DNA sequencing data indicate that SAG contains a potential DNA-binding domain, suggesting that SAG may function as a regulatory protein.