Specimens of cells derived from tumors of the human female genital tract plus normal cells as standards have been divided into aliquots and stained according to acridine orange or pararosanilin:Feulgen procedures. Acridine orange-stained cells were slit-scanned for 535 nm nuclear fluorescence; Feulgen-stained cells were comb-scanned for 580 nm nuclear absorbance. For each specimen examined, the tumor cell:normal cell ratio of mean nuclear fluorescence following acridine orange staining was greater than the tumor cell:normal cell ratio of mean nuclear absorbance following Feulgen staining. The tumor cell:normal cell ratio of mean nuclear fluorescence ranged from 2.3 for a nonkeratinizing squamous cell carcinoma to 3.9 for a keratinizing squamous cell carcinoma. The tumor cell:normal cell ratio of mean nuclear absorbance ranged from 1.4 for a mixed mesodermal sarcoma to 2.3 for a small cell squamous cell carcinoma. These results indicate that the elevated nuclear fluorescence intensity from acridine orange-stained tumor cells cannot be explained solely on the basis of elevated Feulgen:DNA content. An alternative hypothesis, consistent with these results, is that DNA is the principal binding substrate for intranuclear acridine orange and that the DNA of certain tumor cells is more accessible to acridine orange than is the DNA of normal cells.