A number of cell lines, some containing chromosomes with distinctive heteromorphisms, have been flow karyotyped using a single laser flow sorter in an attempt to select those suitable for sorting all human chromosomes individually. Using the non-base-specific DNA stain ethidium bromide, chromosomes 3, 4, 5, and 6 form individual peaks in practically all normal subjects, while the right combination of heteromorphisms enables chromosomes 1, 2, 8, 9, 13, 16, 17, 18, 19, 20, 21, 22, and Y to be sorted separately. Two male cell lines, one containing a duplication and one a deletion of the X, produce flow karyotypes suitable for sorting chromosomes 7 and 8. The use of numerical chromosome abnormalities to enrich the sex chromosomes and the autosomes 18 and 21 is also illustrated. The DNA stain Hoechst 33258 binds preferentially to AT base pairs. Flow karyotypes produced with this fluorochrome separate some chromosomes not well separated with ethidium bromide. Chromosomes 5, 6, 8, 13, 14, 15, 17, and 20, and Y can be sorted individually with Hoechst 33258 with the right combination of heteromorphisms. Using these techniques, all human chromosomes apart from 10, 11, and 12 have been found as individual flow karyotype peaks, suitable for sorting with a high degree of purity.