Chromosomes from antigen stimulated B-cells from spleens of inbred mice have been separated using flow cytometry into 18 distinguishable peaks. Using locus-specific oligonucleotides and fluorescence in situ hybridization to banded metaphase spreads, 15 individual chromosomes were identified: 1, 2, 3, 6, 7, 8, 9, 11, 12, 16, 17, 18, 19, X and Y. The remaining six chromosomes, occurring as pairs in three peaks, 4 with 5, 10 with 13, and 14 with 15, were resolved by flow sorting chromosomes from mice carrying an appropriate homozygous translocation and 4, 5 and 14 have been isolated in this way. This is the first demonstration of how a complete set of mouse chromosome paints can be produced.