We report here the construction of a complete physical map of the chromosome X of yeast Saccharomyces cerevisiae. Fragments resulting from partial Sau3AI digestion of DNA from a diploid strain derived from S288C were ligated to linearized pWE15, a cosmid vector with T3 and T7 promoters. Another library, made in the cosmid vector pOU61 cos, that lacks T3 and T7 promoters, was also used as a source of target clones. Chromosome-X-specific clones were sorted out by hybridization with radiolabelled pulse-field-gel-purified chromosome X as a probe. Then, 254 cosmids were ordered by walking from one to another by hybridization with end-specific T3 or T7 RNA transcripts as probes. The construction was put to the test by hybridization with a battery of chromosome X gene markers, that showed that the physical map and the genetic map were colinear. The validity of the contig was further strengthened by the results of chromosome nested fractionation with meganuclease I-SceI. An EcoRI restriction map of the contig enabled further verification and measurement of the total length of the contig, that was found to be approximately 700 kb in size. In addition to providing a base for the ongoing yeast genome sequencing project, the physical map can be used to map any sequence belonging to chromosome X.