Myosmine is not only one of the minor tobacco alkaloids but is also present in various foods. Therefore, research on myosmine metabolism and activation has been intensified. 3-Pyridylacetic acid, 4-oxo-4-(3-pyridyl)butanoic acid (keto acid), 3-pyridylmethanol, 3'-hydroxymyosmine, and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) have been identified as urinary metabolites after oral administration to female Wistar rats. Although N-nitrosation of myosmine, yielding N'-nitrosonornicotine (NNN) and HPB, was considered as a possible in vivo activation route, the formation pathways of most metabolites could not be explained until now. Therefore, under consideration of its high reactivity due to its imine structure, peroxidation of myosmine seemed to be a promising additional activation pathway. In vitro peroxidation using myosmine (8.9 micromol in 200 microL methanol) with a mixture of hydrogen peroxide (57.6 micromol, 5 microL of a 35% solution) and acetic acid anhydride (106 micromol, 10 microL) already showed high yields of reaction products after 30 min ultrasonic treatment. The product pattern was analyzed by HPLC/UV and GC/MS. Besides unchanged myosmine, 3-pyridylacetic acid, keto acid, 3-pyridylmethanol, HPB, and nornicotyrine have been identified as myosmine peroxidation products. Different product patterns were obtained after 24 h and 4 days due to a time-dependent degradation, formation, and conversion of the reaction products. Therefore, peroxidation reaction of myosmine might explain the in vivo formation of 3-pyridylacetic acid, keto acid, 3-pyridylmethanol, and HPB in rats. In addition, because of acetylating conditions using acetic acid anhydride, N-(4-oxo-4-pyridin-3-yl-butyl)acetamide was rapidly formed during the first 30 min of the reaction.