We have isolated a large noncollagenous glycoprotein, laminin, from a mouse tumor that produces basement membrane. The protein consists of at least two polypeptide chains (Mr = 220,000 and Mr = 440,000) joined to each other by disulfide bonds. Laminin and type IV collagen are major constituents of the tumor. Laminin is distinctly different from fibronectin, another component of basement membranes, in amino acid composition and immunological reactivity. Pepsin digestion of laminin releases a large, cystine-rich fragment which retains most of the antigenicity of the original protein. Immunological studies using purified antibody against laminin show that it is produced by a variety of cultured cells. In addition, these antibodies react with the basement membranes of normal tissues, suggesting that this protein or an immunologically related protein is a constituent of the basement membranes of these tissues.