The localization of the structural gene for human alpha-galactosidase B (= N-acetyl-alpha-galactosaminidase) was investigated by means of man-Chinese hamster and man-mouse somatic cell hybrids. The hybrid clones were analyzed for chromosomes and for a large number of known enzyme markers. The lysates of the hybrid cells were treated with Sepharose-coupled antihuman alpha-galactosidase B and the activity of the adsorbed enzyme was measured on the Sepharose beads as N-acetyl-alpha-galactosaminidase. The results show that the structural gene for human alpha-galactosidase B is situated on chromosome 22, and that there is no structural relationship between human alpha-galactosidase A and human alpha-galactosidase B.