To investigate factors involved in the secretion of protoporphyrin IX (PpIX), a superficial eggshell pigment, from shell gland epithelial cells of Japanese quail, we cultured cells in Ham’s F12 medium with calcium chloride and quail plasma. The addition of hormones (prostaglandin F2α, progesterone, estradiol-17β) to the medium did not change the PpIX concentration in the culture supernatant, but changing the calcium chloride (CaCl₂) concentration did: a lower concentration of CaCl₂ led to a higher PpIX concentration; 0 mM CaCl₂ enhanced the secretion of PpIX from epithelial cells prepared at 5 or 7 mM CaCl₂. The result suggests that a drop in concentration of CaCl₂ mimics the end of shell calcification and stimulates rapid secretion of PpIX in vivo. Bovine serum albumin was almost as effective as quail plasma for PpIX secretion in culture, and would facilitate further study of the mechanism of PpIX secretion.