Confluent monolayers of epithelial cell lines of intestinal derivation may serve as useful models for studies of intestinal epithelial structure and function. Cells of the T84 line appear to be a useful model for studies of Cl- secretion. We now define the fine structure of this cell line, the structural events that occur during monolayer formation, and the influence of plastic versus collagen substrates and permeable versus impermeable substrates on the structural development of these monolayers. When plated on collagen-coated filters at confluency, T84 cells cover the filter surface and lay down basement membrane-like material within 18 h. Such "monolayers" multifocally display areas with many cell layers alternating with areas composed of a single layer of cells. During this stage of development cell polarization is minimal, ultrastructural discontinuities exist that impart low transepithelial resistance, and morphologic features reminiscent of stratified intestinal epithelium during fetal development are present. Within 5 days of plating on collagen-coated filters, true monolayers form. These monolayers have high transepithelial resistance and consist of highly polarized columnar cells with structural similarity to intestinal crypt cells. Cells plated on a plastic substrate are also capable of rapidly producing basement membrane-like material but, unlike those plated on collagen-coated filters, under these conditions the rapid initial spreading of cells over the substrate surface does not occur. As a result, at 5 days monolayers plated on plastic are less uniform than those plated on collagen-coated filters, exhibit areas in which unpolarized flattened cells abound, and multifocally leak macromolecules. Studies using collagen-coated filters made impermeant with glass backing suggest that the enhancing effect of this substrate on monolayer structural development is not simply due to the greater access that basolateral membranes have to nutrient media when grown on permeable supports. This study defines the structure and structural development of this useful model epithelium, and demonstrates the importance of substrate selection.