The use of isoelectric focusing (IEF) to characterize soil humic substances is a technique that appears to be of widespread interest. The use of carrier ampholytes permits the electrophoretie separation in the presence of a pH gradient, but until now the problem of interferences that could occur between the soil organic matter and the carrier ampholytes during the IEF separations have not been recognized. The commercial carrier ampholytes have different chemical structures; thus, interferences with soil humic substances could be different.In this study, two soil extracts were fractionated using the IEF technique in the presence of six commercial carrier ampholytes with a wide pH range (about 3–10). The main results can be summarized as follows:(i) Each ampholyte used had a marked influence on the patterns obtained, and good resolution of the focused bands was obtained only in the presence of some carrier ampholytes (Ampholine and Preblended Ampholine from Pharmacia-LK B and Bio-Lyte from Bio-Rad).(ii) The carrier ampholytes influenced both the number and the apparent isoelectric point of the focused bands.The achievement of a pH gradient that allows electrophoretie runs without formation of complexes and of possible interferences between carrier ampholytes and humic substances is still to be shown. However, the IEF technique is highly reproducible and useful for the characterization of soil humic substances once the experimental conditions and the carrier ampholytes are precisely defined.