Laminins are important for basement membrane structure and function. The laminin alpha2 chain is a major component of muscle basement membranes, and mutations in the laminin alpha2 gene lead to congenital muscular dystrophy in humans and mice. Although the laminin alpha2 chain is prominently expressed in testicular basement membranes, its role in testis has remained unclear. Here, we show that laminin alpha1, alpha2, beta1, beta2, gamma 1, and gamma 3 chains are the major laminin chains in basement membranes of seminiferous tubules. In laminin alpha2 chain-deficient dy(3 K)/dy(3 ASK) mice, lack of laminin alpha2 chain led to concurrent reduction of laminin gamma 3 chain and abnormal testicular basement membranes. Seminiferous tubules of laminin alpha2 chain-deficient dy(3 K)/dy(3 K) mice displayed a defect in the timing of lumen formation, resulting in production of fewer spermatides. We also demonstrate that overexpression of laminin alpha1 chain in testis of dy(3 K)/dy(3 K) mice compensated for laminin alpha2 chain deficiency and significantly reversed the appearance of the histopathological features. We thus provide genetic data that laminin alpha chains are essential for normal testicular function in vivo.