A very rapid separation of H1 and core histones by reversed-phase high-performance liquid chromatography using a Bio-Rad Hi-Pore butyl (C 4) silica-based column in a single run is reported. The histones were dissolved in water containing 0.1% trifluoroacetic acid and fractionated within 20 min by means of a linear gradient system consisting of water-acetonitrile containing 0.1% trifluoroacetic acid. For the detection of histones the eluate was monitored at 210 nm. The identity and purity of eluted proteins were confirmed (1) by acid-urea and Triton-acid-urea polyacrylamide gel electrophoresis, and (2) by comparison with the retention times of pure histone markers. Despite the short elution time, a high resolution of the different histone fractions could be obtained. The eluted histones were recovered in the following order: H1 (LHP), H1 (MHP), H2B, H2A (LHP), H4 plus H2A (MHP), H3 (LHP), and H3 (MHP) (where LHP and MHP refer to less hydrophobic and more hydrophobic histone variants). The reported system is preferable to time-consuming electrophoretic systems for the separation of histones.