Branching morphogenesis in mammary ducts is associated with the expression of a number of proteins. These include laminin 5 and basic fibroblast growth factor (FGF)-2. Both proteins are lost with malignant transformation of mammary epithelium and have causal roles in branching morphogenesis in breast cancer cells in vitro. The in vivo relationships of these proteins with each other and with the loss of branched structures and mammary ductal dedifferentiation are not known. We carried out indirect fluorescence staining on subsets of archived pathologic samples from 55 patients, with a total of 140 pathologic entities, many with multiple stages of dedifferentiation present on the same cut, using antibodies to fibroblast growth factor-2 (FGF-2), fibroblast growth factor receptor-1 (FGFR1), and laminin 5 to determine expression. We also used Western blots to detect laminin 5 expression in MCF-7, T-47D, and MDA-MB-231 cells transfected with vectors constitutively expressing FGF-2 and immunofluorescence staining of matrix proteins deposited by these cells to determine export and accumulation of laminin 5. FGF-2 and laminin 5 expression were found throughout benign and atypical dedifferentiation in mammary tissue samples and were lost primarily with transformation to invasive cancer. FGFR1 was expressed in all cell types. Cancer cells enforced to express FGF-2 did not have detectable laminin 5 on Western blot, but matrix proteins deposited in culture did stain positive, suggesting accumulation of exported laminin 5. Data suggest roles for FGF-2 and laminin 5 in ductal integrity during mammary carcinogenesis, with loss of expression corresponding to loss of ductal structure. In vitro data suggest FGF-2 as causal in laminin 5 expression and export. Down-regulation of FGF-2 during transformation may contribute to loss of laminin 5 expression.