We report the isolation of six cell lines (designated EB cell lines) from cultures of the hypoxanthine guanine phosphoribosyl transferase-deficient (HGPRT-) feeder-dependent embryonal carcinoma cell line PSA4TG12 which have undergone in vitro differentiation, and of clonal derivatives of these lines. Whereas some lines possess quasi-diploid karyotypes similar to that of PSA4TG12, others are markedly aneuploid. Cell line EB26/1 and its clonal derivatives undergo adipogenesis in cultures maintained at confluence; in tumours formed by injection into syngeneic mice they produce muscle-like cells, cartilage and bone in addition to adipose cells. We therefore propose that EB26/1 and its clones are aneuploid derivatives of an uncommitted mesodermal cell. Cell line EB28/5 forms tumours with a histological appearance resembling that of yolk sac carcinoma but does not express biochemical markers characteristic of visceral or parietal endoderm. Cell line EB28/10n has a myoblast-like culture morphology and in tumours is capable of producing muscle-like cells, cartilage and bone. A high specific activity of alkaline phosphatase is present is two of five EB cell lines assayed, and plasminogen activator activity is present in all five. Since the EB cell lines represent populations of cells each expressing a particular subset of the genetic information present in a common ancestral genome, they will be invaluable for studying the developmental regulation of gene expression.