In alphavirus-infected cells the four virus-specific nonstructural proteins (nsP1-nsP4) are located on modified endosomes and lysosomes known as type I cytopathic vacuoles. In this paper, we show that when nsP1 was expressed alone in HeLa cells with the aid of the recombinant T7 RNA polymerase vaccinia (vTF7-3) virus system, it was tightly associated with intracellular smooth membranes. The membrane association may be due to acylation, since nsP1 could be labeled with [3H]palmitic acid in both Semliki Forest virus-infected and nsP1-transfected HeLa cells. Release of the 3H-label by alkaline methanolysis suggests that the palmitate was associated with nsP1 via an ester bond. Pulse-chase experiments done on nsP1-transfected cells revealed that this protein was rapidly associated with the membranes. After synchronizing the synthesis of the nsP1 gene product in transfected cells, nsP1 appeared first at the plasma membrane and thereafter on vesicles, many of which contained the endosomal transferrin receptor marker. Later, nsP1 appeared on large vacuoles, which contained the lysosome specific h-lamp-1 protein. Membrane association of nsP1, and its affinity to endosomes and lysosomes, suggest a role of this protein in the biogenesis of the alphavirus-specific RNA replication complex.