Treatment of F9 teratocarcinoma cells with all trans retinoic acid (RA) causes them to differentiate into two or three morphologically distinct cell types. Whereas the majority of these retinoid-derived cells exhibit properties resembling parietal endoderm, a small percentage of this differentiated cell population manifests properties distinct from the parietal endoderm cell type. The isolation and partial characterization of such a non-parietal endoderm cell line (Dif 5) derived from F9 cells following prolonged (44 days) exposure to 1 μM retinoic acid are described. Unlike the retinoid-induced parietal endoderm-like cell population, which exhibits a dramatic, characteristic morphological change upon treatment with 8-bromo cAMP, Dif 5 cells do not show any morphological change with exposure to this cAMP analog. Dif 5 cells synthesize and deposit an extracellular matrix consisting of several components of Reichert's membrane (fibronectin, laminin, and type IV collagen). This new cell line does not synthesize α-fetoprotein but does secrete plasminogen activator. An interesting property of these cells is their ability to grow in the absence of serum or other hormonal supplements. Yet the Dif 5 cells do exhibit density-dependent inhibition of growth. Unlike the parent F9 cells or parietal yolk sac (PYS-2) cells, these cells do possess specific cell surface receptors for epidermal growth factor (EGF). The growth-arrested Dif 5 cells can be reinitiated to proliferate by the addition of fetal calf serum (FCS) or EGF. The properties of Dif 5 cells determined fail to fulfill all the characteristics described for either parietal or visceral endodermal cells. This raises the possibility that Dif 5 cells might represent an endodermal cell type which is intermediate in differentiation to either parietal or visceral endoderm but which lacks the biochemical signal to complete this stage of differentiation. This new Dif 5 cell line should be of considerable value in studying the modulation of growth requirements and extracellular matrix formation during early embryonic development. © 1983.