The fluorescent characteristics of X (sex) and Y chromatin of human interphase nuclei stained with quinacrine mustard (QM) were studied in fibroblast-like cell cultures derived from several different tissues. X chromatin in female cells was clearly distinguishable from Y chromatin in male cells on the basis of size and fluorescent intensity. The frequency of X chromatin was dependent on cell density in vitro, whereas the frequency of Y chromatin was independent of cell density. The fluorescent intensity of X chromatin in cultured cells was much stronger than that in uncultured cells, such as those of the oral mucosa. The intensity of Y-chromatin fluorescence, however, was nearly the same in both cultured and uncultured cells. Nuclei first stained with QM, photographed, and then restained with thionin and rephotographed showed identical X- and Y-chromatin morphology.