Recent rat studies show different tissue distributions of vitamin B12 (B12), administered orally as hydroxo-B12 (HO-B12) (predominant in food) and cyano-B12 (CN-B12) (common in supplements). Here we examine male Wistar rats kept on a low-B12 diet for 4 weeks followed by a 2-week period on diets with HO-B12 (n 9) or CN-B12 (n 9), or maintained on a low-B12 diet (n 9). Plasma B12 was analysed before, during and after the study. The content of B12 and its variants (HO-B12, glutathionyl-B12, CN-B12, 5'-deoxyadenosyl-B12 (ADO-B12), and methyl-B12 (CH3-B12)) were assessed in the tissues at the end of the study. A period of 4 weeks on the low-B12 diet reduced plasma B12 by 58 % (from median 1323 (range 602–1791) to 562 (range 267–865) pmol/l, n 27). After 2 weeks on a high-B12 diet (week 6 v. week 4), plasma B12 increased by 68 % (HO-B12) and 131 % (CN-B12). Total B12 in the tissues accumulated differently: HO-B12>CN-B12 (liver, spleen), HO-B12<CN-B12 (kidneys), and HO-B12≈CN-B12 (brain, heart). Notably, more than half of the administered CN-B12 remained in this form in the kidneys, whereas HO-B12 was largely converted to the bioactive ADO-B12. Only <10 % of the other cofactor, CH3-B12, were found in the tissues. In conclusion, dietary CN-B12 caused a higher increase in plasma and total kidney B12 but provided less than half of the active coenzymes in comparison to dietary HO-B12. These data argue that HO-B12 may provide a better tissue supply of B12 than CN-B12, thereby underscoring the lack of a direct relation between plasma B12 and tissue B12.