AbstractDeletion of the cortactin gene leads to male infertility. Considering that cortactin is an actin filament (F‐actin)‐binding protein associated with intercellular junctions, we measured changes in the expression and distribution of cortactin and tyrosine phosphorylated cortactin (P‐cortactin) in the seminiferous epithelium of developing and adult mice to address the physiological significance of cortactin to germ cell differentiation. Cortactin was expressed in neonatal and developing Sertoli cells. Cortactin levels decreased early during puberty, while P‐cortactin increased. Cortactin labeling was intense in the basal and apical thirds of the epithelium. Sertoli cell cytoplasmic processes facing spermatogonia, preleptotene spermatocytes, and step 8–13 spermatids were intensely labeled by both cortactin and P‐cortactin. In contrast, the middle region of Sertoli cells exhibited diffuse cortactin labeling but no P‐cortactin. This is consistent with the view that plasma membrane segments facing germ cells are part of the continuum of Sertoli cell junctional complexes that extend over lateral and apical membranes of supporting cells. Moreover, F‐actin and P‐cortactin share a common location in the seminiferous epithelium. The increased P‐cortactin levels detected during puberty may be related to the modulatory effect of cortactin tyrosine phosphorylation on actin assembly at sites of selected Sertoli cell‐germ cell contacts. Cortactin and connexin 43 (Cx43) were physically linked in seminiferous tubule homogenates and their colocalization in the basal and apical thirds of the seminiferous epithelium was stage‐dependent. Our results suggest that cortactin‐Cx43 interaction helps coordinate formation of cell‐to‐cell junctions and organization of the subsurface actin cytoskeleton in specific regions of the epithelium. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.