Pectinase activity was localized at the ultrastructural level in pollen mother cells of tobacco (Nicotiana tabacum L.) during meiotic prophaseⅠto elucidate its role in the biogenesis of secondary plasmodesma (sPD) and cytoplasmic channel (CC). At the leptotene stage the enzyme was mainly present in the cisternae of smooth endoplasmic reticulum (SER) and their derived vesicles, but absent in the Golgi body and Golgi vesicles. Later at the zygotene stage, when sPDs and CCs were actively formed, strong pectinase activity was observed not only in the SER cisternae and their derived vesicles but also in the cell wall, especially in the vicinity of or within both simple and branched plasmodesmata, notably along the middle lamellae, which also characterized the sites of CCs being formed. The presence of exocytotic vesicles containing reaction products suggests that pectinase shares the same excretive pathway as that used by cellulase for its delivery into the wall, i.e. in active form via smooth endoplasmic reticulum (ER) and its derived vesicles by exocytosis. In combination with cellulase, pectinase also promotes the secondary formation of plasmodesmata and CCs by specifically digesting the pectin in middle lamella.