Somatic cells from human females undergo X-differentiation, which curtails expression of most, if not all, of the genes on one X-chromosome. According to the Lyon hypothesis, the designation of whick X will be inactive in eutherian females is random. However, in spite of the obvious biologic importance of X chromosome differentiation, little is known about either the mechanism of this process or the role played by fertilization. Benign ovarian teratomas provide a system for assessing the importance of fertilization in X chromosome differentiation. Biochemical and cytologic data indicate that these teratomas are of germ-cell origin. They are comprised entirely of one of the products of the first meiotic division and are thus parthenogens. In the absence of appropriate recombinational events, ovarian teratomas are consistently homozygous at autosomal loci, even when the host is heterozygous. Analysis of X chromosome replication kinetics provides one additional approach for investigating X-differentiation in individual teratoma cells. We utilized BrdU-dye techniques to study terminal replication patterns in ovarian teratomas and in normal fibroblasts and peripheral lymphocytes from the same individuals. The results confirm that human ovarian teratomas possess a single late-replicating X chromosome. Moreover, the pattern of replication in this X is identical to that in normal fibroblasts, but different from that usually observed in peripheral lymphocytes. Thus, if late replication is an accurate gauge of X-inactivation, the data confirm that X-inactivation can occur without fertilization.