The topographical distribution of type IV collagen, laminin, fibronectin, and the thymocyte differentiation antigen Thy-1 in the basement membrane of the lactating rat mammary gland was investigated. Small cubes of tissue, which had not been subjected to prior fixation or freezing, were incubated with monospecific or monoclonal antibodies to these proteins, and the antibodies were located by an indirect immunoperoxidase staining technique and observed in the electron microscope. The lamina densa stained uniformly with antibodies to type IV collagen and laminin. In addition, both laminin and type IV collagen were present in semiperiodic clusters that traversed the lamina lucida from the cell surface to the lamina densa. Fibronectin was present only in the semiperiodic clusters and not elsewhere in the basement membrane. These clusters were irregularly spaced along the cell surface and heterogeneous in size. It remains to be determined if these three proteins are present in the same clusters. Thy-1 was largely present on the lamina densa and not on the lamina lucida. The Thy-1 staining of the lamina densa occurred in discrete maxima and minima. These maxima occurred in regions adjacent to Thy-1-bearing stromal cells. Thus, the topographical distribution of proteins within a basement membrane varies in a nonrandom manner, and local factors can modify this distribution. We suggest that this topographical variability may play a role in cell recognition and signalling processes that occur across the basement membrane.