The binding of vitamin B(12) by human gastric juice has been found to be pH dependent. Maximum binding occurs between pH 6.5 and 10. Outside this pH range the vitamin B(12)-binding ability of human gastric juice decreases and at pH below 2 or above 12.2 this drops sharply to about 10 to 15% of the maximum. Three commercial hog intrinsic factors have been found to give a similar response to pH changes. The pH-dependent binder in human gastric juice has been shown to be intrinsic factor by the addition of intrinsic factor-blocking antibody. About 10% of vitamin B(12) bound by human gastric juice is not bound by intrinsic factor and is not pH dependent. The reduction in the vitamin B(12)-binding capacity of human gastric juice induced by an adverse pH is reversed by neutralization. The physiological and clinical significance of these observations is discussed and their relevance to various procedures in vitro noted.