Hemidesmosomes (HDs) are specialized cell-substrate junctions with distinct cytoplasmic plaques where intermediate filaments (IFs) are anchored. In our previous work, we described two types of HDs in terms of their molecular constituents, i.e. type I HD and type II HD [Hieda, Y., Nishizawa, Y., Uematsu, J., & Owaribe, K. (1992) J. Cell Biol. 116, 1497-1506]. In the present study we further characterized type II HDs in cultured cells, comparing their composition and function with those of conventional type I HDs. Although the two bovine mammary gland epithelial cell lines, BMGE+H and BMGE-H, were derived from the same tissue, their cell-substrate adhesion properties are markedly different. Immunological examination showed that BMGE-H cells express HD1 and the integrin alpha 6 beta 4 complex but not bullous pemphigoid antigens, while BMGE+H cells express all these components, i.e. the former have type II HDs and the latter have type I HDs. GoH3, a monoclonal antibody to the integrin alpha 6 subunit, inhibited BMGE-H cell adhesion to laminin as a substrate, as also observed for BMGE+H cells. These and electron microscopic results indicate that BMGE-H cells form type II HD-like structures containing HD1 and alpha 6 beta 4 which are associated with IFs. This structure mediates adhesion of the cell to laminin. This is the first demonstration of an adhesion function for type II HDs in cultured cells.