The mechanism of induction of vitamin K (VK) deficiency in newborn babies and antibiotics-treated patients has not entirely been clarified because of the difficulty in preparing the true VK deficient model-animals and the complication in an assay system for VK derivatives and of their metabolites until now. Germfree animal is thought to be an useful tool to establish a primary VK deficiency not caused by VK antagonists etc., because of the lack of their intestinal flora. Germfree (GF) and conventional (CV) ICR/JCL male mice, 12-13 week-old were used in this experiment. VK deficient (K-Def), menaquinone-4 (MK-4) supplemented (MK-4), and VK3 (menadione) supplemented diet (K3) were fed to the mice in both GF and CV states. After 8 days, severe VK deficient symptoms were occurred only in GF-K-Def group, whereas not at all in CV-K-Def group. Both prothrombin time (PT) and activated partial thromboplastin time (APTT) were also prolonged only in GF-K-Def group. From the HPLC analysis of MK-4 content in liver, it was suspected that the content of MK-4 which has been thought to be an active form of VK was not necessarily paralleled with the degree of VK deficiency.