Since alterations in cell replication rate and cell volume distribution are two of the earliest changes seen in the culture of human diploid cells, it was decided to examine the relationship between these parameters. After standardization of the conditions for cell volume measurements (enzyme treatment, temperature and stage of cell growth), a close correlation was observed between cell population doubling time and cell volume in WI-38 cells at various levels of in vitro passage. Cell populations which replicate more slowly (trisomic-21 fibroblasts, fetal skin fibroblasts) also demonstrated a shift to larger cell volumes when compared with control rapidly replicating cell populations at the same level of in vitro passage. Similar shifts to larger cell volumes were produced by reducing serum concentration, decreasing incubation temperature and inhibiting DNA synthesis. Separation of senescent WI-38 cells on the basis of cell volume revealed that the cell fractions with the largest modal cell volume contained the highest percentage of slow or nonreplicating cells. Therefore, an inverse relationship appears to exist between growth rate and cell volume in cultured human diploid fibroblasts.