Mammary branching morphogenesis occurs over a period of weeks deep inside an adipocyte-rich stroma. The adipocytes contain light-scattering lipid droplets that limit the depth of penetration of visible light. Organotypic culture methods were developed to enable high-resolution optical monitoring of branching morphogenesis ex vivo. A challenge has been to identify the best culture conditions to model specific developmental events. We recently demonstrated that collagen I induces protrusive invasion in both normal and neoplastic mammary epithelium. In this study, we observed that the abundance of collagen I fibrils correlated strongly with invasive behaviour, even when the collagen I concentration was identical. We found that the extent of fibril assembly was experimentally manipulable by varying the incubation time at 4°C following pH neutralization. We next tested the capacity of collagen I fibrils to induce invasive behaviour when presented in combination with basement membrane proteins (Matrigel). We found that epithelial organoids in mixed gels of collagen I and basement membrane proteins exhibited more extensive branching morphogenesis but did not initiate protrusions into the matrix. Organoids in pure Matrigel produced many small epithelial buds that were bare of myoepithelial cells. Surprisingly, organoids in mixed gels of collagen I and Matrigel produced fewer epithelial buds, the buds elongated further, and the elongating buds remained covered by myoepithelial cells. Our mixed gels therefore provide a more physiologically accurate model of mammary branching morphogenesis. Our results also suggest that changes in the composition of the extracellular matrix could induce migration of epithelial cells past myoepithelial coverage.