Measurements of the attachment of the PAM 212 line of mouse epithelial cells to various collagen substrates show that these cells adhere preferentially to type IV, basement membrane collagen. Neither serum nor fibronectin stimulated the attachment of these cells (unlike fibroblasts) to type IV collagen. Preincubation of the PAM 212 cells with cycloheximide prevented attachment. Thus these cells do not attach by means of a macromolecule present inserum, but instead synthesize an attachment factor. Extracts of the EHS tumor, which produces an extracellular matrix containing basement membrane components, were tested for their ability to promote attachment to cycloheximide-treated PAM 212 cells. Saline extracts of the tumor stimulated the attachment of the PAM 212 cells to type IV collagen in the presence of cycloheximide. Laminin, a high molecular weight glycoprotein constituent of basement membrane, was purified from the salt extract and was found to be the active species at concentrations as low as 1-5 microgram/ml. When laminin was preincubated on plates coated with either type I, II, III, IV, or V collagen and the plates subsequently washed, high levels of attachment were seen only on type IV collagen-coated plates. Affinity purified antibody directed against laminin inhibited the attachment of PAM 212 cells to a type IV collagen substrate. Laminin appears to be a specific attachment protein for epithelial cells since it did not stimulate the attachment of fibroblasts to type I or to type IV collagen substrates. These data suggest that lamin is produced and utilized by these epithelial cells to attach to basement membrane collagen.