This study was designed to identify the cellular component of the intestinal villus where transcobalamin II (TCII) is synthesized, because this protein provides an essential function in the intestinal absorption of vitamin B₁₂ (cobalamin, Cbl). When a segment of proximal or distal small intestine of the guinea pig is cultured in medium containing [⁵⁷Co]Cbl, TCII-[⁵⁷Co]Cbl appears within 15 min. Northern blot analysis of RNA from both proximal and distal small intestine identified the TCII transcript. In situ hybridization of the distal ileum with ³⁵S-labeled TCII antisense transcript localized grains predominantly in crypts and in the lower third and central core of the villi. Grains were also evident at the base of the enterocytes in close apposition with the vascular network, whereas few grains appeared in the apical region of the columnar cells. This study provides evidence that TCII is constitutively expressed in the intestinal villi where vascular endothelium is abundant. In the distal ileum, where the intrinsic factor (IF) receptor is expressed, after uptake of IF-Cbl and the subsequent binding of free Cbl to TCII synthesized in the villi, the TCII-Cbl complex enters the microcirculation and passes into the portal blood.