During differentiation of isolated Zinnia mesophyll cells into tracheary elements (TEs), lignification on TEs progresses by supply of monolignols not only from TEs themselves but also from surrounding xylem parenchyma-like cells through the culture medium. However, how lignin polymerizes from the secreted monolignols has not been resolved. In this study, we analyzed phenol compounds in culture medium with reversed-phase HPLC, gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry, and found 12 phenolic compounds including coniferyl alcohol and four dilignols, i.e. erythro-guaiacylglycerol-beta-coniferyl ether, threo-guaiacylglycerol-beta-coniferyl ether, dehydrodiconiferyl alcohol and pinoresinol, in the medium in which TEs were developing. Coniferyl alcohol applied to TE-inductive cultures during TE formation rapidly disappeared from the medium, and caused a sudden increase in dilignols. Addition of the dilignols promoted lignification of TEs in which monolignol biosynthesis was blocked by an inhibitor of phenylalanine anmmonia-lyase (PAL), L-alpha-aminooxy-beta-phenylpropionic acid (AOPP). These results suggested that dilignols can act as intermediates of lignin polymerization.