To search for human X-chromosome-specific probes useful for molecular mapping, we studied recombinant clones isolated from a human cDNA library. DNA preparations from 150 randomly selected clones were labeled and annealed to XY and 4XY human DNA, and to DNA from a human-mouse hybrid cell line that had retained only the human X-chromosome (A9/HRBC2). cDNA clones sharing homology with DNA from the X chromosome annealed to A9/HRBC2-DNA and hybridized more intensely to 4XY DNA than to XY DNA. Eleven such clones were identified. Of these, three hybridized only to X chromosomal DNA while the rest also annealed to DNA from one or more autosomes. Chromosomal assignment of the autosomal DNA fragments showed that, in addition to hybridization to X chromosomal DNA, four of the clones hybridized to DNA sequences from chromosome 2 and two clones to chromosome 7. Subregional mapping of the relevant X chromosomal DNA fragments indicated that one clone is homologous to DNA sequences located at Xp21-Xp22, whereas the others are located in the telomeric region of the long arm. The cDNA clones were used to search for restriction fragment length polymorphisms. Several restriction-site polymorphisms were detected. Some corresponded to variants of X chromosomal DNA sequences while others were from autosomes such as chromosomes 2 and 7.