Using isoelectric focusing in thin-layer agarose gel (AGIF) with the narrow pH range of 4.5-5.4, a high resolution of esterase D (ESD) isozyme banding patterns has been achieved. Some variant phenotypes which could not be distinguished from common ESD types by conventional electrophoresis have shown different patterns after AGIF. The IEF method permitted the distinction of two further variants in the ESD system, tentatively named ESD Rehren and ESD Ravensburg. We recommend, therefore, that for the classification of ESD phenotypes a high resolution IEF technique should be used.