The compartmentalization of peroxidase isoenzymes related to lignin biosynthesis was studied in the cell walls and the cell wall-free spaces of several woody and non-woody plant species. The results illustrate a specific compartmentalization of peroxidase isoenzymes in cell walls compared with that found in vacuoles. Cell wall peroxidase isoenzymes may be classified into three main groups: one with acidic pI (APrx), and two with a basic pI (BPrx LpI and BPrx HpI). While basic peroxidases are constitutively expressed, acidic peroxidases are normally developmentally regulated. This developmental regulation and the greater efficacy in the oxidation of coniferyl alcohol shown by acidic peroxidases suggest that this isoenzyme group plays a key role in lignin biosynthesis, although some contribution to this process by basic peroxidases cannot be ruled out.Peroxidase (EC 1.11.1.7) is the enzyme responsible for the linking and cross-linking of monolignols during the biosynthesis of lignins in the plant cell wall. As may be expected from its specific role in lignin biosynthesis, peroxidase is mainly located in the cell wall, although other subcellular localizations have been reported. Over the last few years, considerable information has been accumulated about the subcellular localization of peroxidase isoenzymes (7). However, it remains unclear whether the findings concerning isoenzyme localization in a few plants can be generalized and rationalized. Combined histochemical, cytochemical and biochemical studies are necessary to rationalize some concepts which are only just beginning to be understood.To cast light on this question, we have studied peroxidase isoenzyme localization in lupin (Lupinus albus) (2-4), grapevine (Vitis vinifera) (5, 6), pepper (Capsicum annuum) (7, 8), lettuce (Lactuca sativa) (9), catharanthus (Catharanthus roseus) (10), oats (Avena sativa) (Rojas, M.C.; Ros Barcelo, A., unpublished data) and aleppo pine (Pinus halepensis) (Ros Barcelo, A., unpublished data). Electron microscope cytochemistry, vacuum infiltration and subcellular fractionation, together with protoplast and vacuole isolation techniques, show that in all the studied plant materials both basic (BPrx, pi > 7.0) and acidic (APrx, pi < 7.0) peroxidase isoenzymes are located in the cell wall free spaces, probably in equilibrium with those bound to the cell 84