Hypotonic treatment of amnion fluid cell-nuclei was found to enhance demonstration of Y chromatin and of autosomal fluorescence in interphase cell-nuclei following quinacrine mustard staining. In contrast, visualization of the Barr body was not improved by this treatment. Nuclear sex determination on cellular material from 200 samples of amniotic fluid was unequivocal in 197 cases; in only 3 cases were the findings uncertain. Therc were no erroneous determinations. In one instance maternal cellular tissue was observcd in smear preparations. Nuclear sex determination presents a simple method of supplementing and substantiating prenatal chromosomal diagnosis.