Newly synthetized derivative of bile acid, sodium salt of 3α, 7α-dihydroxy-12-oxo 5β cholanic acid (monoketocholanate) expressed a good characteristic as intranasal transport enhancer of xenobiotics.The aim of our sudy was to explore if it has an influence on bile metabolism and to measure its concentration in blood and bile after intravenous and intranasal administration. The experiment was performed in vivo on adult male Wistar rats. The determination of monoketocholanate (MKCh) in rats blood and bile, was carried out by high-performance liquid chromatography (HPLC), on an HP ODS2 column, using methanol/acetonitrile/acetate buffer as mobile phase. Absorbances were measured at 210 nm.Blood samples were taken from the prepared right axillary artery in 0, 1, 5, 10, 20, 30, 60, 90, 120 and 180 minutes from the beginning of the experiment. Bile was collected in a half an hour intervals,during the three hour period. The results showed that MKCh changed the amount of excreted bile depending on the way of application. Intranasal application increased the bile volume and the MKCh concentration, both in blood and bile compared to the intravenous application (p<0.05). Distributionm of MKCh through animal organism depends on the way of application of the substance, which probably determines its caracterisation as the transport promotor of applied xenobiotics. HPLC has proved as aa relatively simple, fast and effective method for the determination of synthetic bile acid,MKCh in these biological materials.