A clonal cell with an acinar cell phenotype, which was induced by 5-azacytidine treatment of a neoplastic human salivary intercalated duct cell line, was cultivated in the presence of 1 alpha,25-dihydroxyvitamin D3. Morphological changes occurred; large cells that were polygonal or round in shape and had numerous vacuoles in their cytoplasm appeared in the treated cells, whereas the same concentration of 1 alpha,25-dihydroxyvitamin D3 did not affect the morphology of the parental cells. Major alterations, such as expression of type II collagen, alpha and beta chains of S-100 protein, and sulfated proteoglycans, were observed in these cells with a phenotype similar to chondrocytes. After the removal of 1 alpha,25-dihydroxyvitamin D3 from the culture, the treated cells returned rapidly to the phenotype of the untreated cells. These findings indicate that the reversible differentiation into chondrocyte-like cells of a human salivary acinar cell line occurs in growth medium containing 1 alpha,25-dihydroxyvitamin D3.