Apical ectoplasmic specialization (ES) is a testis-specific hybrid cell/cell actin-based adherens junction and cell/matrix focal contact anchoring junction type restricted to the interface between Sertoli cells and developing spermatids. Recent studies have shown that laminin γ3, restricted to elongating spermatids, is a putative binding partner of α6β 1-integrin localized in Sertoli cells at the apical ES. However, the identity of the α and β chains, which constitute a functional laminin ligand with the γ3 chain at the apical ES, is not known. Using reverse transcription-PCR and immunoblotting to survey all laminin chains in cells of the seminiferous epithelium, it was noted that α2, α3, β1, β2, β3, and γ3 chains were found in germ cells, whereas α1, α2, α4, α5, β1, β2, γ1, γ2, and γ3 chains were found in Sertoli cells, implying that α3 and β3 are the plausible laminin chains restricted to germ cells that may be the bona fide partners of γ3. To verify this postulate, recombinant proteins based on domain G of α3 and domain I of β3 and γ3 chains were produced and used to obtain the corresponding specific polyclonal antibodies. Additional studies have demonstrated that the laminin α3, β3, and γ3 chains indeed are restricted to germ cells at the apical ES, co-localizing with each other and with β1-integrin. Furthermore, co-immunoprecipitation studies have confirmed the interactions among laminin α3, β3, and γ3, as well as β1-integrin. When the functional laminin ligand at the apical ES was disrupted via blocking antibodies, such as using anti-laminin α3 or γ3 IgG, this treatment perturbed adhesion between Sertoli and germ cells (mostly spermatids), leading to germ cell loss from the epithelium. More important, a transient disruption of the blood-testis barrier was also detected.