Chinese hamster cell deficient in hypoxanthine-guanine phosphoribosyl transferase(HPRT) was fused with normal human female lymphocyte. Chinese hamster-human cell hybrid clones retaining human inactive X chromosome were selected. The hybrid cells were treated with the de(hypo)methylating agent 5-azacytidine(5-AzaC) and tested for derepression of the three inactive human X-linked genes. Hprt, glucose-6-phosphate dehydrogenase (G6pd), and phosphoglycerate kinase (Pgk). Depression of Hprt gene was detected by pulse-labeling and histochemical methods. The frequency of derepression of Hprt gene after the treatment with 5-AzaC for one cell cycle was 21-fold greater than that observed in the untreated hybrid cells. Data using electrophoretic method showed that the hybrid cells in which Hprt was derepressed also expressed human G6PD and PGK, but that the frequencies of derepression of Pgk and G6pd genes were independent. These results support that the derepression of the inactive human X-linked genes is affected by 5-AzaC-induced de(hypo)methylation of DNA.