Cytoplasmic Ca2+ concentration of epithelial cells from 14-day embryonic chick duodena decreased during 72 h of organ culture to a value 54% of that found at 17 days in vivo. The ability of cells to maintain a constant Ca2+ concentration when challenged with high extracellular calcium was also significantly reduced. Addition of 1 microM hydrocortisone during culture restored both parameters of Ca2+ homeostasis to that of 16-day uncultured duodena, and rise in cytoplasmic Ca2+ was significant within 4 h of hormone treatment. Thyroxine influenced epithelial Ca2+ similarly, but to a lesser degree and only after 48-72 h of culture. These data indicate that glucocorticoids, and possibly thyroid hormones, influence the development of calcium homeostasis in intestinal epithelium.