The effect of three different substrates, laminin, fibronectin and reconstituted basement membrane, on isolated mouse pre-Sertoli cells maintained in vitro has been investigated. Cultures were monitored on a daily basis by phase contrast microscopy, and processed for light and electron microscopy at the end of the culture period. Extra-cellular matrix components have been found to influence Sertoli cell differentiation: both fibronectin and laminin promoted cell adhesion and differentiation, though laminin cultures showed poor viability. Pre-Sertoli cells cultured on reconstituted basement membrane showed the highest degree of differentiation, developing a polarized epithelial phenotype and forming cord-like aggregates; such cultures provide a model system for the investigation of factors involved with gonadal differentiation.