Hybrid cell lines isolated after fusions between Chinese hamster E36 cells and normal human white blood cells were analyzed for human beta-galactosidase isoenzymes and for human chromosomes, especially 3, 12, and 22, the candidates for bearing a beta-galactosidase locus. Results of neuraminidase treatment of the cell lysates and immunological studies showed that in man two structural beta-galactosidase loci are present and can be assigned to chromosomes 3 and 22. No correlation was found between the expression of human beta-galactosidase and the presence of human chromosome 12.