Flow cytometry can be used for the detection of aberrations in DNA composition and DNA content of human chromosomes. We demonstrate here the magnitude of DNA content differences among normal chromosomes of 20 healthy individual donors and the clinical applications of the standardized bivariate flow karyotype. Peak positions were determined from Hoechst 33258 (HO) VS Chromomycin A3 (CA3) flow karyotypes of individuals, and the values of the relative chromosomal DNA content were normalized to the peak position constituting chromosomes 9-12, to which a value of 100 arbitrary units (a.u.) was assigned. The mean values and standard deviations that were HO and CA3 fluorescence intensity for each chromosome were calculated. The range of standard deviation is 1.43 to 6.21 (CA3) and is 1.22 to 4.28 (HO). The standard deviations for each chromosome did not overlap; therefore, it was decided that the standardized bivariate flow karyotype was suitable to use for clinical applications. Further study using fluorescence in situ hybridization for chromosome 22 that was observed for extra peak accompanied with normal peak in the flow karyotype of clinical material did not suggest more positive data. This suggests that the peak-position difference between chromosome 22 homologues may be a result of a combination of satellite and heterochromatic-region size differences.