An interspecific cell hybrid was constructed between a temperature-sensitive mutant cell line of mouse FM3A cells (FT20-M6, a 6-thioguanine-resistant derivative of tsFT20) that has heat-labile DNA polymerase alpha and a human diploid fibroblast derived from a patient with the fragile X syndrome. After extensive segregation of the human chromosomes, a hybrid clone, named M6-39-11, was obtained that contained an X chromosome as the only human chromosome, was able to grow at the nonpermissive temperature and contained human DNA polymerase alpha. These data strongly suggest that the functional human gene for DNA polymerase alpha is located on the X chromosome.