The preparation of stable chromosome suspensions and flow cytometric sorting of both the Y sex chromosome of the white campion, Melandrium album, and the deleted Y chromosome of an asexual mutant, 5K63, is described. The principle has been to maintain transformed roots in vitro, synchronise and block mitosis, reduce cells to protoplasts, and lyse these to release chromosomes. Such in vitro material, unlike many cell suspensions, showed a stable karyotype. Factors critical to producing high-quality chromosome suspensions from protoplasts include osmolality of isolation solutions and choice of spindle toxin and of lysis buffer. Agrobacterium rhizogenes transformed young growing root cultures were synchronised at Gl/S with 50 pM aphidicolin for 24 h and released to a mitotic block with 30 pM oryzalin for 11 h. Protoplast preparations from such tissue routinely had metaphase indices reaching 15%. Suspensions of intact metaphase chromosomes, with few chromatids, were obtained by lysing swollen mitotic protoplasts in a citric acidldisodium phosphate buffer. Except for the presence of clumps of autosoma1 chromosomes near the X and Y chromosome zones, monoparametric histograms of fluorescence intensities of suspensions stained with 4',6-diamidino-2-phenylindole showed profiles similar to theoretical flow karyotypes. Two types of Y chromosomes, one full-length and one partially deleted (from the asexual mutant), could be sorted at 90% purity (21-fold enrichment of Y). These results are discussed in the context of sex determination and differentiation in higher plants. The availability of purified individual chromosomes facilitates the study of molecular properties of eukaryotic genomes. This is based mainly on the enrichment of potentially interesting DNA as compared with the total genomic content. The construction of chromosome-specific DNA libraries greatly facilitates the isolation and ordering of DNA sequences located on these chromosomes. Flow cytometry offers the possibility to isolate specific chromosomes. Individual chromosomes differing sufficiently in DNA content (generally proportional to relative length) and/or base pair composition are represented as separate peaks in the flow karyotype (18).