The effect of colchicine, Nocodazole, and dibucaine on the assembly of Semliki Forest virus was investigated. Colchicine, Nocodazole, and dibucaine reduced the production of extracellular virus by 75 to 90%. Lumicolchicine had no effect on virus growth. Other control experiments showed no effect by these drugs on the incorporation of [3H]leucine into material precipitated by trichloroacetic acid. Colchicine (100 micron) disrupted the microtubles of the baby hamster kidney cells (BHK-21), whereas dibucaine did not alter microtubule polymerization. The stage of virus assembly inhibited by colchicine and dibucaine was studied by experiments with [3H]-leucine or [35S]methionine. At various times after addition of one of these drugs, the incorporation of the labeled precursors into viral proteins associated with fractions enriched for endoplasmic reticulum or plasma membrane from the cell was evaluated. The results clearly show that the envelope and nucleocapsid proteins of the virus move to the plasma membrane of the cell where they accumulate. The studies strongly suggest that the cytoskeletal system is involved in the final stages of morphogenesis of Semliki Forest virus from the plasma membrane.