To investigate the possible role(s) of cytoskeletal elements in desmosome assembly we have studied the effects of cytostatic drugs on the assembly of desmosomes in MDCK epithelial cells. We showed previously [Pasdar et al.: Cell Motil. Cytoskeleton 23:201-213, 1992] that selective disruption of microtubules has no effect on desmosome assembly. Here, we have treated MDCK cells with cytochalasin B and a combination of cytochalasin B and nocodazole and analysed the effects of desmosome assembly. Immunofluorescence analysis of MDCK cultures following drug treatment indicated complete disruption of actin microfilaments and disorganization of cytokeratin intermediate filaments. Biochemical analysis of newly synthesized desmosomal membrane core glycoproteins as well as the cell adhesion protein E-cadherin revealed no effect of these drugs on the kinetics of synthesis, intracellular processing, or transport to the plasma membrane either in the presence or absence of cell-cell contact. However, morphological analyses revealed a significant disruption in the spatial organization of desmosomal proteins and E-cadherin. Drug treatment in the absence of cell-cell contact resulted in the disruption of the normally observed homogeneous punctate staining pattern and appearance of aggregate staining. Induction of cell-cell contact in these cultures resulted in redistribution of some of the aggregate staining to the plasma membrane. In contrast to control cultures, significant amount of intracellular staining was retained for all desmosomal proteins. Biochemical analyses of turnover rates of newly synthesized desmosomal proteins indicated a significant decrease in metabolic stability of these proteins while the turnover rate of E-cadherin was not significantly different among control and drug-treated cultures. Taken together, these results suggest that intact actin and cytokeratin filaments are necessary for the stability, efficient assembly, and spatial organization of the junctional components at the membrane. The regulatory role of cytokeratins and actin filaments in assembly and stability of desmosomes on the plasma membrane is discussed.