During 24 h of organ culture, rabbit gastric fundic mucosal explants maintain constant tissue levels of intrinsic factor (IF) while steadily secreting this glycoprotein into culture medium. Mucosal explants thus generate in one day an average of 1.8 pmol of new IF per milligram mucosal protein, an amount corresponding to 70% of IF present in explant tissue. Cultured explants also incorporate [35S]methionine into tissue IF and secreted IF at a constant rate. Histamine combined with isobutylmethylxanthine stimulates explants to release IF into the culture medium, but tissue levels of IF are diminished and specific activities of tissue and secreted IF remain the same. Fluorograms of 35S-labeled proteins generated by cultured explants fail to show cobalamin-binding precursors or breakdown products of IF. These findings complement previous morphological documentation of IF synthesis by gastric mucosa. Histamine appears to stimulate IF secretion without altering IF synthesis.