We adapted a method for evaluating monoclonal antibody specificity toward isoforms and examined 29 alpha-fetoprotein (AFP) antibodies from Hybritech. These antibodies were separated by affinity electrophoresis on agarose-containing erythroagglutinating phytohemagglutinin (PHA-E) followed by immunoblotting onto nitrocellulose. Under these conditions, AFP separates into four subfractions: NR (nonreactive with PHA-E), WR (reactive weakly), RS2 (strongly reactive), and RS1 (very strongly reactive). When polyclonal goat anti-AFP control was used as the test antibody, the distribution of amniotic fluid AFP was approximately 50%, 31%, 12%, and 7% for NR, WR, RS2, and RS1, respectively. The distribution for these same bands from the serum of a patient with hepatocellular carcinoma was 39%, 31%, 30%, and 0%; from the serum of a patient with embryonal carcinoma, it was 29%, 38%, 0%, and 33%, respectively. Twenty-six monoclonal antibodies, including the two used in the Hybritech Tandem-E assay for AFP, showed similar distribution, while the remaining three antibodies showed no reactivity to any of the AFP antigens in this system. Although we were not able to demonstrate any specificity of these antisera toward any single AFP isoform, use of this affinity electrophoresis system provides a model for studying isoform specificity of other AFP antibodies and other antibody-antigen systems.