Laminin was recently characterized as being a major non-collageneous protein of the basement membrane matrix produced by a mouse tumor. It was extracted from the tumor matrix with neutral buffer and purified under non-denaturing conditions. Rabbit and guinea pig antisera raised against laminin or a large pepsin fragment P1 of laminin showed strong binding to both laminin and peptide P1 but only a weak reaction with reduced and alkylated laminin. The major antigenic determinants of laminin were located in a disulfide knot, comprising one third of the molecule, which resisted degradation by pepsin or cyanogen bromide. Minor antigenic determinants shared by the native and reduced protein could also be identified. The data were interpreted as showing that laminin consists of conformationally rigid as well as more flexible domains. Absorption studies with mouse kidney homogenate indicated that authenic basement membranes contain a protein immunologically identical to laminin. Tissues from other species contain a related protein which exhibits partial cross-reaction with mouse laminin. Together with immunofluorescence data the findings demonstrate that laminin, like type IV collagen, occurs in most basement membranes of the body.