Ecto-5'-nucleotidase activity (5NT) was measured on whole cells of 26 human x Chinese hamster hybrids. Concordance analysis showed 100% correlation between enzyme activity and inheritance of human chromosome 6. This observation was confirmed by a segregation analysis in which cells of a hybrid containing chromosome 6 were stained by indirect immunofluorescence for HLA Class 1 antigen and sorted by a fluorescence-activated cell sorter (FACS). Cells in the HLA- compartment were cloned and expression of HLA and 5NT was determined. Of nine clones, three were HLA-, 5NT- and six were HLA+, 5NT+, supporting the linkage of 5NT to chromosome 6.