T he mechanism of bacterial uptake of vitamin B₁₂, the spectrum of microorganisms capable of such uptake, and the factors involved were the subject of this study. Bacterial uptake of vitamin B₁₂ was found to be at least a two stage process. A primary uptake phase which was rapid (1 min or less), pH dependent, nontemperature dependent, did not require viable organisms and was insensitive to either the metabolic inhibitor dinitrophenol or to the sulfhydryl inhibitor N-ethyl-maleimide. Protein denaturation (formalin treatment or autoclaving) abolished all B₁₂ uptake. This primary uptake phase is thought to represent adsorption to binding or “receptor” sites on the cell wall. Second stage uptake was slower, pH and temperature dependent, required living bacteria, and was abolished by either dinitrophenol or N-ethyl-maleimide. This phase is dependent upon metabolic processes and may reflect transfer of B₁₂ from surface “receptor” sites into the bacterial cell. Although differences among organisms were observed in total 1 hr uptake, number of surface “receptor” sites, and relative avidities for B₁₂, all organisms except Streptococcus fecalis shared the two stage mechanism. Two Gram-positive organisms. Bacillus subtilis and Group A streptococcus, demonstrated the highest 1 hr vitamin B₁₂ uptake values; Gram-negative bacteria required 2,000-10,000 the number of organisms for comparable uptake. Binding constants (K_m) varied from 5.05 ±1.67 × 10^-10M for B. subtilis to 6.18 ±3.08 × 10^-9M for Klebsiella pneumoniae which approximate the Km for human intrinsic factor (0.38 × 10^-10M). Competition between bacteria and intrinsic factor for vitamin B₁₂ may be inferred from the similarity of these constants.These observations suggest that a variety of enteric and nonenteric organisms, not requiring exogenous B₁₂, may play a role in the pathogenesis of the vitamin B₁₂ malabsorption found in the intestinal bacterial overgrowth syndromes.