Localization of the enzymes involved in lignification is essential to better understand the dynamic aspect of lignin biosynthesis in the cell, as well as to better understand the regulation of lignification in woody plants. Lignification has three steps: 1) biosynthesis of the monolignols, 2) transport and secretion of monolignols, and 3) dehydrogenative polymerization of the monolignols. Though most of the enzymes involved in lignification have been identified, their localization in the cell and cell wall remains equivocal. To obtain more detailed information, we investigated the localization of enzymes involved in lignification within the cell and the cell wall in poplar trees using immunocytochemistry. Immunolabeling of phenylalanine ammonia-lyase, caffeate O-methyltransferase, 4-coumalate:CoA ligase, and cinnnamyl alcohol dehydrogenase was localized in the differentiating xylem. These enzymes were particularly abundant during secondary wall formation. Immunolabeling was observed on the polysomes and in the cytosol of the cells during secondary wall formation, indicating that these enzymes are synthesized in the polysomes and released in the cytosol. The synthesis of monolignols might occur in the cytosol. Immunolabeling of peroxidase was also localized in the differentiating xylem, particularly during secondary wall formation. The labeling, however, was observed in the rough-endoplasmic reticulum, the Golgi apparatus, and the plasma membrane, indicating that peroxidase is synthesized in the r-ER, transported to the Golgi apparatus, and localized at the plasma membrane by fusion of the Golgi vesicles to the membrane. The most interesting feature of the present results is that the enzymes involved in monolignol synthesis are separated from the enzyme involved in polymerization of monolignols in the cell by the membrane. Monolignols passing through the plasma membrane are polymerized dehydrogenatively in the cell wall in the presence of peroxidase and hydrogen peroxide.