Technical Abstract: Phylloquinone (PK) is converted into menaquinone-4 (MK-4) via side chain removal-addition. Stable isotope use is an effective approach to identify the tissue location of this conversion, which is currently unknown. Following a 14 d PK-deficient diet, male Fischer 344 rats (8 mo; n=15) were fed 1.6 mg deuterium-labeled PK (L-PK) per kg diet for 0 (C), 1d (PK-1) and 7 d (PK-7). Both L-PK and deuterium-labeled MK-4 (L-MK-4) were detected in tissues in PK-1 and PK-7, although results varied. Whereas some tissues had an overall increase in MK-4 in response to L-PK, total brain, testes and fat MK-4 concentrations did not. In contrast, L-MK-4 concentrations increased in all three tissues. The deuterium label was found only on the L-MK-4 naphthoquinone ring, confirming the need for side chain removal for formation of MK-4. Labeled menadione (L-MD) was detected in urine and serum in PK-1 and PK-7, confirming its role as an intermediate. A Caco-2 cell monolayer model was used to study the role of the enterocytes in the conversion process. Neither MK-4 nor MD was detected in Caco-2 cells treated with PK. However, when Caco-2 cells were treated with MD, MK-4 was formed. Similarly, MK-4 was formed in response to MD-treated kidney cells. These data demonstrate that MK-4 is the predominant form of vitamin K in multiple tissues, but there appears to be a tissue-specific regulation for the conversion of PK to MK-4.