Rat liver microsomes can oxidize glycerol to formaldehyde. This oxidation is sensitive to catalase and glutathione plus glutathione peroxidase, suggesting a requirement for H₂O₂ in the overall pathway of glycerol oxidation. Hydrogen peroxide can not replace NADPH in supporting glycerol oxidation; however, added H₂O₂ increased the NADPH-dependent rate. Ferric chloride or ferric-ATP had no effect on glycerol oxidation, whereas ferric-EDTA was inhibitory. Certain iron chelators such as desferrioxamine, EDTA or diethylenetriaminepentaacetic acid, but not others such as ADP or citrate, inhibited glycerol oxidation. The inhibition by desferrioxamine could be overcome by added iron. Neither superoxide dismutase nor hydroxyl radical scavengers had any effect on glycerol oxidation. With the exception of propyl gallate, several antioxidants which inhibit lipid peroxidation had no effect on formaldehyde production from glycerol. The inhibition by propyl gallate could be overcome by added iron. In contrast to glycerol, formaldehyde production from dimethylnitrosamine was not sensitive to catalase or iron chelators, thus disassociating the overall pathway of glycerol oxidation from typical mixed-function oxidase activity of cytochrome P450. These studies indicate that H₂O₂ and nonheme iron are required for glycerol oxidation to formaldehyde. The responsible oxidant is not superoxide, H₂O₂, or hydroxyl radical. Cytochrome P450 may function to generate the H₂O₂ and reduce the nonheme iron. There may be additional roles for P450 since rates of formaldehyde production by microsomes exceed rates found with model chemical systems. Elevated rates of H₂O₂ production by certain P450 isozymes, e.g., P450 IIE1, may contribute to enhanced rates of glycerol oxidation.