For logarithmically growing cultures of the human diploid cell lines WI-38 and WI-26, there is an exponential increase in the fraction of cells not incorporating [3H]dT under conditions of continuous labelling. Thymidine uptake, phosphorylation and incorporation into DNA can be correlated with cell proliferation. In addition, determination of the labelling index is reproducible within relatively broad limits of thymidine concentrations and specific activity. The plateauing phenomenon of the curve expressing percent-labelled nuclei versus time, which occurs in populations with less than 100% cycling cells, is largely due to radiation damage to the cells. The results of these studies provide insight into the population dynamics of aging fibroblast-like cell cultures. More importantly, however, the measurement of labelling index as described can be used as a reproducible and quantitative measure of the age of the diploid cell culture.