Degraded macromolecular lignin, which was isolated from the effluents of commercial pulp processing and known to inhibit early development in marine organisms, was separated and characterized using several polyacrylamide gel electrophoresis (PAGE) techniques. This lignin-derived macromolecule (LDM), when subjected to native PAGE and stained with alcian blue, appeared as a single band. On sodium dodecyl sulfate (SDS)-PAGE, LDM appeared to consist of two subcomponents with apparent molecular weights of 11 and < 1 kDa. When subjected to isoelectrofocusing--PAGE of pH 3-9, LDM consisted of two major bands in the basic region of the gel, with less distinct banding in the more acidic region. Two-dimensional PAGE of LDM indicated that the higher molecular weight subcomponent corresponded to the more basic constituents, while the lower molecular weight subcomponent corresponded to acidic constituents. When the two subcomponents of LDM were isolated from SDS gels by electroelution and assessed for their effects on successful fertilization and early development, the higher molecular weight subcomponent possessed most of the inhibitory activity. This is the first report of the application of a variety of electrophoretic techniques to both structurally and biologically characterize lignin-derived macromolecules.