A series of human probes with unique sequences has been isolated from a recombinant phage library constructed with DNA obtained from a human-hamster hybrid cell line. This cell line contained the X chromosome as the only human component. For 18 of these probes, a human X-chromosome origin has been confirmed and they have been regionally assigned by a combination of techniques: dosage studies utilizing DNA from human fibroblasts carrying X-chromosome duplications and deletions; the presence or absence of hybridization to digested DNA from hybrid lines carrying fragments of the X chromosome; and in situ hybridization to metaphase chromosomes. The use of dosage as a means to regionally assign probes significantly improves resolution of the X chromosome.