Immunocytochemical techniques were used to study the distribution of fibronectin, type IV collagen (collagen-IV), and laminin in four different stages of mouse blastocyst development. Immunoreactivity for collagen-IV and laminin is present in a granular pattern inside the inner cell mass (ICM) cells in stage 1 blastocysts, while these blastocysts are negative for fibronectin. Fibronectin immunoreactivity appears extracellularly under the trophectoderm (TE) in stage 2 blastocysts, in the form of homogeneously distributed dots, and/or fibrils located preferentially close to cell boundaries. It is followed by the appearance of both collagen-IV and laminin immunoreactivity in patches on the basal side of the TE in stage 3 blastocysts. These patches are initially localized under the central region of TE cells, thus in a location clearly different from that of fibronectinpositive fibrils. As development proceeds the collagen-IV- and laminin-positive patches become larger, covering, by stage 4, an extensive portion of the inner lining of the blastocoel. Fibronectin-positive material is still present in a fibrillar form in stage 3 blastocysts, but is generally reduced to thin strands by stage 4. These results indicate that fibronectin is independent of the mouse blastocyst basement membrane, but may play a transient role in cell adhesion during its deposition. In addition, the results suggest that the ICM plays a major role in the production of collagen-IV and laminin, while the basal surface of TE cells is the primary site of basement membrane assembly.