Abstract Bioassay-directed fractionation of organic compounds was performed on an organic solvent extract of a contaminated estuarine sediment from Black Rock Harbor, Connecticut, using the new mutagenic bioassay, Mutatox™. Chemical fractionation methods of the sediment extract included silica-gel-column chromatography (SGCC), followed by C18 reverse phase HPLC. Compound identification was performed using GC-MS. Mutatox analyses indicated that four of the eight HPLC fractions contained mutagenic or epigenetic compounds. GC-MS analyses detected known mutagenic polycyclic aromatic hydrocarbons (PAHs) in two of the four mutagenic fractions. GC-MS analyses of the other two mutagenic fractions (one operational blank and one sediment fraction) showed no known mutagenic compounds. SGCC and HPLC, combined with the Mutatox bioassay, were useful in reducing both the number and the complexity of fractions analyzed by GC-MS. The Mutatox bioassay is a promising tool for bioassay-directed fractionation.