7-Ketocholesterol (7KC) and 7β-hydroxycholesterol (7βOH) are oxygenated metabolites of cholesterol. They are implicated in manipulation of lipid transport, development of oxidative stress & apoptosis in the vascular wall. Recent studies have demonstrated that oxysterols are inter-converted by 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), better known for its role in amplifying local glucocorticoid concentrations. Inhibition of 11βHSD1 protects against atherosclerosis, but it is uncertain whether this reflects altered glucocorticoid, or oxysterol metabolism. Our study aimed to compare 11βHSD1 metabolism of 7KC & 7βOH with that of corticosterone. Kinetics of oxysterol and glucocorticoid metabolism were determined using mouse recombinant 11βHSD1 protein, liver microsomes and kidney homogenates from C57Bl/6 and 11βHSD1^-/- mice (male, 9wks, n=6). Oxysterols and glucocorticoids were quantified by HPLC-UV/radiodetection and GCMS. In microsomes, 11βHSD1 catalysed dehydrogenation of 7βOH to 7KC (Km = 3.5μM; Vmax = 32.6 pmol/min/mg); the affinity for 7βOH was 10 fold less than that for corticosterone (B) (Km = 0.314μM Vmax = 4.654 nmol/min/mg). Dixon plots and global fit analysis have shown these substrates to be mutual competitive inhibitors (R2=0.79),and this confirmed previous Lineweaver-Burk transformation data of IC₅₀ for 7βOH inhibitor of 2.20 ± 9.853μM with B range [10^-9-2M]. 11βHSD1 specificity for this reaction was confirmed by lack of similar metabolism in microsomes from 11βHSD1^-/- mice.Furthermore, the inability of 11βHSD2 to catalyse this reaction was established in renal homogenates. Recombinant 11βHSD1 was also able to catalyse NADPH dependent reduction of 7KC to 7βOH,although dehydrogenation was the preferred direction (Km 7βOH = 327.5μM; Km 7KC = 1.2mM). Similarly, the catalytic constants for substrates suggested higher turnover of 7KC than 7βOH substrate Kcat 7KC = 1.4 pmol/sec, Kcat 7βOH=0.114pmol/sec).The 7α-epimer (7αOH), was not a substrate for dehydrogenation, nor was it generated by reduction of 7KC. In silico modelling of enzyme:substrate interactions, based on published 11βHSD1-crystallography data, suggested more favourable binding between Y183-S170-NADP⁺ catalytic triad of 11βHSD1 and the 7βOH-epimer as opposed to the 7αOH. These data confirm that murine 11βHSD1 catalyses the metabolism of specific 7-oxysterols as well as glucocorticoids. This suggests that elevated circulating concentrations of oxysterols in dyslipidaemia, may impair glucocorticoid action, while varying glucocorticoid levels may influence oxysterol action.This interaction may be crucial in understanding the influence of corticosteroids and oxysterols in the pathophysiology of cardiovascular disease.