Abstract The isolated perfused bovine udder is an in-vitro model, which maintains bovine udder skin with an isolated vasculature in a viable state. Using this in-vitro model, the percutaneous absorption and metabolism of isosorbide dinitrate (ISDN) was studied. The organ was perfused with gassed Tyrode solution for up to 6 h. A region of udder skin was treated topically with 60 mg ISDN as a spray, 60 mg ISDN as an ointment and with 120mg ISDN as a microemulsion patch of 30 cm2. Spray and ointment were applied onto a skin region of 400 cm2. The concentrations of ISDN and its metabolites isosorbide-2-mononitrate and isosorbide-5-mononitrate were measured in perfusate fractions by capillary column gas-liquid chromatography with electron capture detection. Following topical administration of the different formulations, ISDN as well as its metabolites were detected in the perfusate fractions, thus demonstrating that ISDN is metabolized by the udder skin in-vitro. A maximum amount of ISDN was absorbed after administration as a spray followed by ointment and microemulsion (5, 2·5 and 1·8 μmol total organic nitrate, respectively). In contrast, the ISDN flux per cm2 skin was significantly higher after administration of the microemulsion (64·4 pmol cm−2 min−1 for the microemulsion compared with 21·9 and 10·2 pmol cm−2 min−1 for spray and ointment).