Epithelial cell differentiation is regulated by specific combinations of growth factors, hormones, and extracellular matrix (ECM). How these divergent signals are integrated is largely unknown. We used primary cultures of normal human bronchial epithelial cells (NHBEs) to investigate mechanisms of signal integration. In defined, serum-free media, NHBEs undergo mucosecretory differentiation only when grown in the presence of retinoids and on the appropriate substratum (collagen gels). We identified the retinoic acid receptor β (RARβ) gene as an early marker of NHBE differentiation. In contrast to immortalized cell lines, in NHBEs strong retinoid-induced RARβ transcription occurs only when cells are grown on collagen gels, and it requires new protein synthesis and a cis-acting element that maps outside the known RARβ promoter elements. NHBEs grown on collagen gels exhibit reduced epidermal growth factor (EGF)-induced Raf, MEK, and mitogen-activated protein kinase (MAPK) activity. This correlates with a specific inability to achieve high levels of p66^SHC tyrosyl phosphorylation and association of p66^SHC with GRB2, despite high levels of EGF receptor (EGFR) autophosphorylation. Notably, inhibition of EGFR or MEK/MAPK activation replaces the ECM requirement for RARβ induction. Our results strongly suggest that a key mechanism by which specific ECMs facilitate retinoid-induced mucosecretory differentiation of NHBEs is by restricting the level of EGFR-dependent MEK/MAPK activation evoked by autocrine and/or paracrine EGFR ligands.