Chromosome G-banding analysis of two human mammary carcinoma cell lines, Elco and MCF-7, showed the existence of two X chromosomes in both cell lines. To determine the state of activity of the X chromosomes, a methylation-sensitive restriction endonuclease, HpaII, was used to distinguish the active X from the hypermethylated, inactive X chromosome with a probe for the phosphogalactokinase locus by Southern blot hybridization. DNA digested with the restriction enzymes PstI and BstXI showed a band at either 1.05 or 0.9 kilobases. After HpaII digestion, a 50% reduction in intensity was observed in the female controls, whereas total reduction of the band was observed for the tumor cell lines and the male control. This indicates the absence of an inactive X and the presence of only active X chromosomes in the mammary carcinoma cell lines and the male control. To investigate the mechanisms involved in the alteration of the X chromosome composition and activity, restriction fragment length polymorphism analyses of seven additional X chromosome markers (L1.28, DX13, p52A, pX65H7, L782, pA13.RI, and pXG-12) were performed on the DNA isolated from the tumor cells and controls. Heterozygosity for at least one of the seven markers was detected in the six female controls whereas only homozygosity was detected for each marker in the tumor cell lines and the male control. These results indicate that the two active X chromosomes identified in each of the two tumor cell lines are identical, resulting from duplication or nondisjunction of the active X and loss of the inactive X chromosome.