Crossed immunoelectrofocusing comprises isoelectric focusing in the first dimension in combination with electrophoresis into antibody-containing gel in the second dimension. Isoelectric focusing on a thin layer polyacrylamide gel permits better comparison of a number of different samples in the same run compared to isoelectric focusing in polyacrylamide rods. Strict control of several parameters during electrofocusing, slicing of the acrylamide gel and transfer of the strips to an agarose gel were found to be important factors in obtaining reproducible results. Attempts to mould acrylamide strips into agarose gels were abandoned when a more convenient way of achieving acrylamide-agarose contact, referred to as the laying-on method, was devised, which gave highly reproducible results. In this technique the acrylamide strip was turned upside down after electrofocusing and applied to the surface of an antibody-containing agarose gel. Crossed immunoelectrofocusing was used to study different preparations of diphtheria toxin from three laboratories. All preparations contained toxin with pronounced microheterogeneity as evidenced by fused immunoprecipitation patterns. A possible explanation for the microheterogeneity of diphtheria toxin is that 'nicked' toxin may contain up to six different molecular species.