Three new cell lines (NE, ME, LRD) were cloned from mouse-embryo-derived teratocarcinomas and characterized on the basis of developmental, ultrastructural, and cytochemical criteria as nullipotent embryonal carcinoma (EC), pure parietal yolk sac (PYS) carcinoma and mixed parieto-visceral yolk sac carcinoma respectively. Cell lines NE and ME were composed of a monomorphous cell population; however, the morphology of ME was growth-medium-dependent. LRD was composed of a heterogeneous cell population and formed embryoid bodies. NE secreted soluble laminin, osteonectin, entactin and fibronectin but did not form visible pericellular matrix. ME formed pericellular matrix which was composed of laminin and entactin, but did not contain fibronectin. The LRD cells formed pericellular matrix which was composed of laminin, entactin and fibronectin. Whereas laminin from ME and LRD reacted with polyclonal antibodies and a monoclonal antibody to parietal yolk sac laminin, the laminin from NE cells was unreactive with the monoclonal antibody. Osteonectin was found in the supernatant of LRD and ME, but could not be demonstrated immunohistochemically in the extracellular matrix. We conclude that some extracellular matrix components, such as laminin and fibronectin, are produced not only by yolk sac carcinoma cells but by nullipotent EC as well, although the latter do not assemble them into extracellular matrix. Laminin produced by EC is immunochemically different from laminin secreted by yolk sac carcinoma. The extracellular matrix produced by mixed parieto-visceral yolk sac carcinoma is different from the matrix laid down by the pure PYS in that the latter does not contain fibronectin. The lack of osteonectin in the extracellular matrix of yolk sac carcinoma cells indicates that not all polypeptides secreted by these cell lines are incorporated into the extracellular matrix. The new cell lines described in this paper differ with regard to their capacity to form extracellular matrix and secrete its various components. Hence they could be used for further studies of basement membrane assembly in vitro.