We previously demonstrated an inverse relationship between the G1 volume of human diploid fibroblast-like (HDFL) cells obtained from foreskin tissue and clonal replicative potential. O n the basis of these results, we suggested that one process underlying in vitro senescence is a progressive increase in the mean cell volume of successive progeny within clonal lineages. We now report that the size of HDFL cells, as well as of chick embryo fibroblasts, can be increased in the virtual absence of cell division by culturing at low density and at low serum concentration (0.1-1 .(I%). Consequent to an increase in cell size, the replicative potential of the cells is reduced to the level of later-passage cells of similar size. By clonal analysis, the populations of enlarged cells contain up to three times as many nondividing cells as do controls. In the enlarged populations, the proportion of cells producing attenuated clones (four or fewer progeny) increases by about 30%, whereas the proportion of cells yielding >:I2 cells declines by a similar percentage. These observations lead us to propose that replicative potential may be limited by cell size, which in turn may be regulated by a kinetic relationship between cellular growth and cell division cycles.