A method for staining of alpha-galactosidase with the synthetic substrate alpha-naphthyl-alpha-galactopyranoside after isoelectric focusing on gel slabs has been devised. Depending on the method used for cell extraction, at least seven isozymes could be detected in cell extracts of cultured fibroblasts from normal individuals. Thermal treatment revealed that both heat-stable and heat-labile isozymes occur in normal fibroblasts. The heat-labile isozymes were not detected in cells from Fabry hemizygotes and thus truly reflect products of the alpha-gal A locus. Three heat-stable isozymes observed in normal individuals were also found in Fabry heterozygotes and hemizygotes and are presumably determined by the alpha-gal B locus. The remaining isozymes were stained very weakly in the hemizygotes and were heat-stable. The relation of these isozymes to the A or B locus is uncertain. After treatment with neuraminidase the alpha-gal A isozymes could not be detected and one of the alpha-gal B isozymes appeared broader. The isozyme pattern observed in heterozygotes was almost identical to the normal one.