Enzymatic and topochemical aspects of lignification were studied in a Pinus radiata D. Don cell culture system that was induced to differentiate tracheary elements and sclereids with lignified secondary cell walls. The activities of the lignin-related enzymes phenylalanine ammonia lyase (PAL; EC 4.3.1.5) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) increased concomitantly with cell differentiation, indicating that the increase in enzyme activity was related to lignification of the cell walls and was not induced by stress. This result also indicates that PAL and CAD are suitable markers for tracheary element differentiation in coniferous gymnosperms. To further characterize lignification in this cell culture system, cellular UV-microspectrophotometry and thioacidolysis were employed. Typical UV-absorption spectra of lignin were obtained from the secondary cell walls of the tracheary elements and sclereids and from the compound middle lamella connecting differentiated cells, and the presence of lignin was confirmed by thioacidolysis. Certain aspects of lignin topochemistry in the cell walls of the tracheary elements were similar to cell walls of P. radiata wood, such as the high lignin concentration in the compound middle lamella connecting adjacent cells and the lower lignin concentration in the secondary cell walls. Therefore, the P. radiata cell culture system appears to be well suited to study the formation of lignified secondary cell walls in coniferous gymnosperms.