Background information: Delta-like proteins 1 and 2 (DLK1, 2) are NOTCH receptor ligands containing epidermal growth factor-like repeats, which regulate NOTCH signalling. We investigated the role of DLK and the NOTCH pathway in the morphogenesis of the submandibular salivary glands (SMGs), using in vitro organotypic cultures. Results: DLK1 and 2 were present in all stages of SMG morphogenesis, where DLK1 inhibited both NOTCH activity and SMG branching. The addition of NOTCH inhibitory agents, either soluble DLK1 (sDLK1) or N-[N-(3, 5-difluorophenacetyl-L-alanyl]-S-phenylglycine t-buthyl ester (DAPT), to the SMG culture medium did not affect the rate of cell proliferation, but induced a strong reduction in SMG branching, increased epithelial apoptosis, and impaired innervation of the epithelial end buds by local parasympathetic ganglion neurons. SMG innervation could be restored by the acetylcholine analog carbachol (CCh), which also rescued cytokeratin 5 (CK5+)-expressing epithelial progenitor cells. Despite this, CCh failed to restore normal branching morphogenesis in the presence of either sDLK1 or DAPT. However, it improved recovery of branching morphogenesis in SMGs, once DLK1 or DAPT were removed from the medium. Conclusions: Our data suggest that DLK1 regulates SMGs morphogenesis and parasympathetic nerve fibre outgrowth through inhibition of NOTCH signalling. Research article: DLK1 and 2 proteins are expressed in submandibular salivary gland (SMG) development, and DLK1 inhibits NOTCH activity. SMG branching was reduced in the presence or NOTCH inhibitory agents, either soluble DLK1 (sDLK1) or DAPT, and impaired innervation of the epithelial end buds. SMG innervation was restored by the acetylcholine analog carbachol, which also rescued cytokeratin 5 (CK5+)-expressing epithelial progenitor cells. It improved recovery of branching morphogenesis in SMGs, once DLK1 or DAPT were removed from the medium. © 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.