Two new lines of human endometrial carcinoma (HEC) cells, one from an adenocarcinoma and one from a highly metastatic serous papillary carcinoma, were established in culture. Structural and morphologic properties of these cells at early passage were compared with those of cultured normal human endometrial epithelial (NHEE) cells. For these studies, cells were grown on a conventional plastic surface or on an extracellular matrix substrate (Matrigel), and examined by transmission electron microscopy and immunofluorescent light microscopy. The HEC cells appeared morphologically similar on plastic and Matrigel, whereas the NHEE cells showed significantly greater epithelial morphologic differentiation on Matrigel than on plastic. On extracellular matrix, the morphologic differences observed between HEC cells and NHEE cells were primarily of an architectural nature, which may be in part explained by differences between NHEE and HEC cells in the arrangement of actin microfilaments and cytokeratin intermediate filaments. Furthermore, HEC cells displayed extensive networks of vimentin intermediate filaments, which were absent from the NHEE cells. These observations support the hypothesis that architectural deregulation is a prominent feature of endometrial carcinoma, and that cytoskeletal alterations may uncouple HEC cell ultrastructural morphology from the influence of extracellular matrix.