Simple epithelial cells are polygonal in shape, polarized in an apical- basal orientation, and organized into closely adherent sheets, characteristics that result from a variety of cellular specializations and adhesive proteins. These characteristics are lost when the epithelia transform during embryogenesis into mesenchymal cells or after neoplasia into invasive carcinoma cells. Of the syndecan family of transmembrane heparan sulfate proteoglycans, simple epithelia produce predominately syndecan-1, which is found at basolateral surfaces and within adhesive junctions. To elucidate the function of this syndecan-1, normal murine mammary gland epithelia were made deficient in syndecan-1 by transfection with an expression vector containing the syndecan-1 cDNA in the antisense configuration. Several independently derived clones of stable transfectants contained the antisense cDNA in their genome and expressed the antisense transcript. These grew either as epithelial islands of closely adherent polygonal cells, identical to both the parental cells and the vector-only control transfectants, or as individual elongated fusiform cells that invaded and migrated within collagen gels, like mesenchymal cells, but were anchorage-independent for growth. The clones that retained epithelial characteristics were moderately deficient in cell surface syndecan-1 (greater than 48% of control levels) but did not differ from control cells in expression of β1-integrins and E-cadherin, or in F-actin organization. However, the clones of fusiform cells were severely deficient in cell surface syndecan-1 (less than 12% of control levels) and showed rearranged β1- integrins, markedly reduced E-cadherin expression, and disorganized F-actin filaments, but retained mammary epithelial markers. Therefore, depleting epithelia of cell surface syndecan-1 alters cell morphology and organization, the arrangement and expression of adhesion molecules, and anchorage-dependent growth controls. Thus, cell surface syndecan-1 is required to maintain the normal phenotype of simple epithelia.