We are especially interested in understanding the mechanisms that regulate the morphogenesis of Wolffian/epididymal duct (WD) because disruptions to epididymal function may also arise as a consequence of abnormal development. Elongation and coiling of the WD are not trivial events but must be highly coordinated with its specialized function of providing a unique luminal fluid microenvironment that is so important for sperm maturation. Our previous study showed that mediolateral intercalation of epithelial cells was a major driver of ductal elongation and regulated by protein tyrosine kinase 7 (Ptk7), a member of the planar cell polarity non-canonical Wnt pathway. However, findings from this study also suggested that mesenchymal cell radial intercalation contributed to ductal morphogenesis. Therefore, we tested the hypothesis that Ptk7 regulated mesenchymal cell radial intercalation via regulation of the deposition of the extracellular matrix (ECM) and intracellular activities of myosin and RAC1. Using a conditional knockout approach, we found that loss of Ptk7 resulted in abnormal assembly of nephronectin, laminin, and collagen IV at the basement membrane coupled with fibrosis-like deposition of fibrilla collagen in the interstitium. When in-vitro-cultured WDs were treated with collagenase IV, the degree of cross-linking of fibrilla collagen was reduced, which resulted in reduced elongation and coiling, and an expanded cyst-like duct. Furthermore, the activity levels of RAC1 and myosin II decreased in the Ptk7 knockout mesenchyme compared to controls. When WDs were treated with RAC1 inhibitor NSC23766, mesenchymal fibrilla collagen was disassembled, and WD elongation was significantly reduced. Our data suggest that Ptk7 regulates the interaction between the ECM and mesenchymal intracellular myosin and RAC1 activities. This interaction allows for cell movement/intercalation resulting in an epididymal duct of the correct length and size, which is important for normal male fertility. Supported by the Eunice Kennedy Shriver NICHD/NIH grant RO1 HD069654.