Septum formation and mitosis were investigated by light and electron microscopical techniques as well as through the completion of inhibitor experiments in the unicellular desmid Xanthidium armatum (Bréb.) Rabenh. In untreated cells, numerous endoplasmic reticulum (ER) cisternae permeated the mitotic apparatus and secretory vesicles, and ER formed a band in front of the linearly growing septum, indicating the predetermined direction of septum growth. Under the influence of cytochalasin D (CD), the vesicle/ER band lost its proper orientation, which led to a malformed septum wall; moreover, abnormal septum branches could potentially have developed. Whereas the septum of an untreated cell only grew at its edge, the CD-induced branches (also with a vesicle/ER band in front) represented additional growing zones. These observations indicated that actin filaments were involved in establishing, maintaining, and orienting the “preforming” vesicle/ER band and, thus, the later septum. Latrunculin B (LB) had more severe effects on septum formation than did CD. Only small accumulations of septum material were found at the septum edge, and no aberrant growth of the septum occurred in LB-treated as in CD-treated cells. This could be explained by the more rapid disturbance of all actin-driven processes after LB treatment, which was assumed, because even low concentrations of the drug rapidly inhibited cytoplasmic streaming.