To determine the status of heterochromatin while it was undergoing DNA synthesis, human female fibroblasts were exposed for a brief period of time (three minutes) to tritiated thymidine and immediately fixed. Autoradiography demonstrated that the sex chromatin body retains its heterochromatic form during DNA replication. This was confirmed by the observation that the percentage of sex chromatin positive cells was not significantly different for cells in the S period of the cell cycle as compared with cells in the G1 or G2 period. The nature of the labeling differentiated three types of sex chromatin bodies: those that were unlabeled; those that labeled with a grain density equivalent to euchromatin (early labeled); and those that labeled with a grain density much greater than that of the surrounding euchromatin (late labeled). Of 138 cells, the sex chromatin was unlabeled in 34%; early labeled in 45%; late labeled in 21%. These fractions were multiplied by the duration of the S phase of DNA synthesis (7.5 hours) determined on the same culture of cells. This demonstrated that the inactive X chromosome does not begin DNA replication until 2.5 hours after the onset of replication of euchromatin. It then continues with an intensity of labeling equivalent to euchromatin for 3.4 hours, and continues replication for 1.6 hours after most euchromatin has ceased replication. The total duration of replication of the inactive X chromosome was approximately five hours, compared with over six hours for euchromatin. The implications of these observations in relation to histones, chromosome coiling, DNA and RNA synthesis, number of replicons in the nucleus and sex chromatin negative cells are discussed.