We isolated interspecific hybrids between normal human leukocytes and a Chinese hamster ovary cell line that has mutations in three genes, leuS, emtB, and chr, all of which are linked to chromosome 2. The conditionally lethal mutation in the leuS gene in this cell line affects leucyl-tRNA synthetase and renders the cell line nonviable at 39 degrees C. The mutation in the emtB locus alters ribosomal protein S14 and results in the cell line being resistant to the protein synthesis inhibitor, emetine, while the mutation in the chr locus renders the cells resistant to sodium chromate. The interspecific hybrids were selected at 39 degrees C so that they were required to retain and express the human leuS gene. Ten out of ten such heat-resistant hybrids also expressed the human emtB and chr genes. Segregants selected as having lost the human emtB gene simultaneously lost the human chr and leuS genes as well. The linkage relationship between these three genes has thus been conserved during the evolution of the human and Chinese hamster genomes. All three genes were localized to human chromosome 5. Furthermore, our results indicate that the ribosomal protein product of the human emtB gene is incorporated into functional ribosomes in place of the human corresponding Chinese hamster protein, raising several interesting questions concerning the coordinate regulation of genes encoding ribosomal proteins in mammalian cells.