Background: During kidney development, the embryonic collecting duct (CD) epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. It is unknown by which molecular mechanism the different cell types arise. We have experimental evidence that the electrolyte environment is involved in the process of terminal cell differentiation. Methods: Embryonic CD epithelia from neonatal rabbit kidneys were microsurgically isolated and maintained in gradient perfusion culture for 13 days under serum-free conditions. Controls were maintained in the same medium (Iscove’s modified Dulbecco’s medium; IMDM) on basal and luminal sides. Experimental series were performed with IMDM only on the basal side, while on the luminal side IMDM with increasing concentrations of NaCl was used. Finally, the development of principal and intercalated cell features was registered by immunohistochemical labeling with markers specific for adult CD cells. Results: Immunohistochemical markers show that the differentiation pattern is quite different when the embryonic CD epithelia are cultured in IMDM only as compared with specimens kept in IMDM supplemented with 3–24 mmol/l NaCl on the luminal cell side. First signs of changes in development were seen when low doses of 3–6 mmol/l NaCl were added. Conclusions: We conclude that facultative protein expression in embryonic CD epithelium is influenced by the electrolyte environment and starts to be upregulated after administration of unexpectedly low doses of 3–6 mmol/l NaCl added to the luminal perfusion culture medium and increases in a concentration-dependent manner.