Subcutaneous fibrosarcomas were induced chemically in mosaic mice heterozygous at the Pgk-1 locus on the X-chromosome. The normal tissues of these animals displayed two electrophoretically distinct isozymes of phosphoglycerate kinase-1-[(PGK-1) EC 2.7.2.3]. The tumors were carefully dissected and analyzed with respect to PGK-1 isozymes. A combination method of multiple sampling of the primary tumor and establishment of tissue culture cell lines from the tumor was utilized. A total of 82 samples was studied from 10 tumors developed in 9 mice. Twenty cell lines were established from 9 tumors; 6 of the tumors were shown to contain neoplastic cells of exclusively PGK-1B activity, 3 of the tumors were shown to contain neoplastic cells of exclusively PGK-1A activity, and 1 tumor contained neoplastic cells with both PGK-1A and PGK-1B activities. The tumor with both A and B activities was hourglass shaped, and the pattern of activity from multiple samples of this tumor suggests that it was formed by the coalescence of a PGK-1A and a PGK-1B tumor. The data presented are concordant with results from chimeric animals and spontaneous tumors in humans and indicate that these tumors display the behavior of clonal growths.