Embryonic mouse submandibular epithelia initiate branching morphogenesis within two days when embedded in Matrigel and stimulated by members of the epidermal growth factor family. However, it is unknown whether the end buds further branch over longer culture periods, and whether saliva-producing cells differentiate there. In the present study, we cultivated three major (submandibular, sublingual and parotid) salivary epithelia from 13-day embryos for 14 days in mesenchyme-free cultures. All epithelia continued to grow and branch to form numerous acinus-like structures in medium supplemented with neuregulin 1, fibroblast growth factor 1, and insulintransferrin-sodium selenite. Alcian blue staining to detect mucous cells showed that each epithelium differentiated via three distinct modes, as seen in normal development, although the staining intensities were weaker than in normal development. RT-PCR analysis of the amylase gene showed that no epithelia expressed amylase after 14 days of culture, which is inconsistent with the fact that only parotid epithelium does so at postnatal day 7 during normal development. These results suggest that cytodifferentiation progresses to a lesser extent in mesenchyme-free cultures than in vivo.