The complex topological association of Sertoli cells and spermatogenic cells in the testis suggests the existence of cell surface adhesion molecules that regulate cellular interactions within the seminiferous epithelium. The recent report of N-cadherin mRNA expression in the mouse testis implies the involvement of this known adhesion molecule in testicular cell binding. Accordingly, here we report that (1) N-cadherin is found on the surface membranes of rat spermatogenic cells and on Sertoli cells, and (2) that N-cadherin is a partial mediator of Sertoli cell-germ cell adhesion as tested in an vitro cell-cell binding assay. Antiserum directed against the N-cadherin cell adhesion recognition sequence was used for Western blot anlaysis of purified plasma membranes from Sertoli cells and from spermatogenic cells. Both membrane preparations exhibited reactivity at an appropriate M(r) of about 130 kDa. In addition, immunofluorescence assays demonstrated that both germ cells and Sertoli cells were labeled by anti-N-cadherin. Finally, the antiserum was included in a cytometer-assisted cell-cell binding test to determine its inhibitory ability. The antiserum consistently reduced specific testicular cell-cell adhesion by 30%-50%. This is the first demonstration that antibodies directed against the cadherin cell adhesion recognition sequence are capable of inhibiting cell-cell interactions. Pre-incubation of either rat Sertoli cells or spermatogenic cells alone was sufficient to achieve statistically significant inhibition of intercellular adhesion. We conclude, therefore, that N-cadherin is expressed by both Sertoli cells and spermatogenic cells and that N-cadherin is one of a number of regulatory molecules mediating local cellular associations in the mammalian seminiferous tubule.