In many plant species, suberin lamellae are deposited in the cells of the endodermis. Some cells near the xylem poles tend to remain without these lamellae and are known as “passage cells”. To develop a three-dimensional picture of the pattern of suberin lamella deposition, procedures were developed to isolate large pieces of onion endodermis and to stain their suberin lamellae for various types of microscopy. Sudan red 7B proved satisfactory with white-light, Fluorol yellow and berberine with epifluorescence, and Nile red with confocal laser scanning microscopy (CLSM). Lamella deposition commenced in a few cells 10 mm from the root tip and then proceeded in a scattered manner. However, lamella deposition in more cells (seen 125–155 mm from the root tip) resulted in the formation of longitudinal files of cells with lamellae alternating with files of passage cells. In older regions (255–285 mm from the tip), almost all cells had deposited suberin lamellae, leaving only a few passage cells. At higher magnification, three-dimensional reconstructions of CLSM images of endodermal suberin lamellae revealed that they are perforated by pores all along the roots. Such pores could serve as points for water and ions to enter the cytoplasm of the endodermis.