Cell-wall (CW) development in the desmid Penium margaritaceum (Ehrenb.) Bréb. was studied using immunofluorescence labeling of living cells with the monoclonal antibodies (mAbs) JIM5 and JIM7, which recognize unesterified and methyl-esterified homogalacturonan (HG), respectively. During cell expansion, HG was secreted in a high-esterified form at a narrow band, called the HG secretion band or HGSB, at the isthmus or the polar tip of a daughter semicell. As newly secreted HG is displaced outward on the cell surface, deesterification and subsequent calcium (Ca2+)-complexing occurred to yield a rigid covering. HG secretion and CW/cell expansion were reversibly inhibited by dark, brefeldin A (BFA), and incubation in 0.24–0.36 M sucrose but were not altered by treatment with actin/microfilament drugs. The HGSB was detected near the nucleus during most cell-cycle events. Centrifugation displaced the nucleus away from the HGSB, but HG synthesis was not affected. HGSB activity was preceded by, and coordinated with, Calcofluor labeling, which suggests that cellulose production in CW/cell-expansion sites was critical to expansion control. In many first-cell-division products, asymmetric patterning of HG was noted in the CW. These asymmetric patterns most likely were a result of timing mechanisms and displacement of the nucleus-HGSB during the cell cycle.