Relative nuclear DNA content and nuclear area were determined in fast growing, karyotypically normal, fibroblastic cell cultures derived from five human females. In two cultures H3 thymidine autoradiographic and photometric determinations were combined on the same nuclei. This allowed the separation of nuclei into three groups: those with the lowest mean DNA values which did not take up H3 thymidine are in the G1 period (2d group); those with intermediate DNA values which did take up thymidine are presumably synthesizing DNA and are in the S period; those which have double the mean 2d group DNA value and did not take up thymidine are a mixture of nuclei in the G2 period with some true tetraploids 4d group). The overall frequency of sex chromatin positive nuclei in the cultures studied ranges from 64 to 78. Sex chromatin positive nuclei occur with about the same frequency in 2d and S nuclei but are somewhat more frequent in 4d nuclei. In three cultures the relative DNA content of sex chromatin was determined. Sex chromatin of 4d nuclei contain about twice as much DNA as those of 2d nuclei; the DNA content of sex chromatin of S nuclei is between that of 2d and 4a nuclei but closer to the mean 2d value. Sex chromatin can be identified in labelled S nuclei. These observations clearly indicate that the condensed, sex chromatin forming X chromosome does not despiralize completely during DNA replication as some authors have suggested. The DNA content of sex chromatin positive and negative nuclei is identical within each nuclear group. The areas of sex chromatin positive 2d group nuclei are larger than for negative nuclei, but this difference is only significant in one of the cultures where a large number of nuclei were measured. It is concluded that failure of the X chromosome to condense in some nuclei of females is not related to DNA
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