A temperature-sensitive mutant of Syrian hamster cells, AF8 was fused with simian-virus-40-transformed Lesch-Nyhan fibroblasts; LNSV, in the presence of beta-propiolactone-inactivated Sendai virus. The AF8 cells grow well at 33.5 degrees but are arrested in mid G1 period when shifted to 39 degrees. The LNSV cells are deficient in hypoxanthine guanine phosphoribosyltransferase. The hybrid clones were selected in hypoxanthine-aminopterin-thymidine medium at 39 degrees. A total of 20 clones was isolated and karyotyped. All clones contained most of the hamster chromosomes and one to eight human chromosomes. The preferential retention of human chromosome 3 was observed in 100% of the metaphases of all clones. In nine of the clones, the only human chromosome present was chromosome 3. The results indicate that human chromosome 3 is responsible for conferring to the hybrid cells the ability to grow at nonpermissive temperature for AF8 cells, i.e., the ability to overcome the G1 block. These findings, together with other reports in the cell cycle may be regulated by genes located on different chromosomes.