Electrofocusing could be usefully employed to characterize humic substances, but careful investigation is needed because of possible interactions of humic substances with carrier ampholytes. In the present work, three fractions of humic substances which originated bands in different parts of the pH gradient during electrofocusing were isolated by preparative electrophoresis in phosphate buffer pH7 from 0.1 M Na4P2O7extracts of the A horizon of a lithic Rendoll (Fusine) and of leonardite. Infrared spectra and E4/E6ratios showed distinct compositional differences among different fractions, while corresponding fractions from humic substances of different origin displayed evident similarities. In both cases, infrared spectra showed a progressive increase in carboxyl groups and a corresponding decrease in ketonic and quinonic carbonyl groups, passing from fractions with the lowest electrophoretic mobility and highest apparent isoelectric points (H) to those with the highest electrophoretic mobility and lower apparent isoelectric points (L). Electrophoretic fractions from the protorendzina soil were also analyzed by pyrolysis gas chromatography (Py-GC). Results confirmed information obtained by spectroscopic methods and showed that the H fraction had the most complex composition with covalently linked polysaccharide and protein derived parts and was composed of poorly humidified materials. © 1990 Williams & Wilkins.