The genetic linkage relationships of the human glycosphingolipid fl-galactosidases were determined using human-mouse somatic cell hybrids. A new method was devised for the estimation of human galactosylceramide, lactosylceramide, and GMt-ganglioside [3-galactosidase activities in the presence of their mouse counterparts, which takes advantage of the reproducible specific activity of lysosomal hydrolases under a given set of culture conditions and is based on differences in both pH optima and sensitivity to chloride ion. Human and mouse chromosomes were identified by their characteristic banding patterns obtained after quinacrine staining, and the optimum glycolipid p-galactosidase activity was determined.for three different substrates. A ratio ,was defined for each activity which was the specific activity at the human pH optimum divided by the specific activity at the mouse pH optimum. Linear regression analysis was used to test .for concordant segregation between pH ratios for each enzyme and the frequency of occurrence of different human chromosomes in the manmouse somatic hybrid clones. The results obtained from two independent series of hybrid clones indicated that human [3-galactosidase activities consistently segregated with human chromosome 12 in these somatic cell hybrids.