A series of 18 Chinese hamster × human somatic cell hybrid subclones segregating human chromosomes were analyzed by two-dimensional gel electrophoresis to determine the chromosomal location of the human genes responsible for the expression of specific proteins. Two independent methods, side by side comparison and double label autoradiography-fluorography, were used to identify the human proteins that were unambiguously resolved from those of the Chinese hamster (CH). Of the 83 human spots resolved, 22 were determined to be associated with nine specific chromosomes or chromosome regions by correlation with quantitative cytogenetic analysis of the hybrid clones. An additional five spots were associated with three different chromosomes that had identical patterns of segregation. A total of 12 resolvable polypeptides of apparently human origin were present in all 18 hybrid subclones and may be coded for by one of the nine human autosomes that were also consistently present. Future investigations using a variety of available methods to determine the identity of the polypeptide spots will permit the mapping of genes for specific enzymes and for other classes of proteins that are not detectable by the commonly used zymogram methods.