We have created somatic cell hybrids between the temperature-sensitive mouse cell line tsA1S9 and human cell lines in order to localize the human gene (A1S9T) complementing the cell cycle defect of the murine line. Segregation of the human X chromosome is completely concordant with growth at the nonpermissive temperature. Hybrids retaining the X chromosome are temperature-resistant, whereas those without a human X are temperature-sensitive. Further hybrids made using human cell lines with X-autosome translocations indicate that the A1S9T gene is located on the short arm of the human X chromosome.