The expression of the middle-component (M) RNA of cowpea mosaic virus was studied by means of in vitro translation. In both the wheat germ extract and the rabbit reticulocyte lysate, M RNA was translated into two overlapping polypeptides of 95 and 105 kilodaltons. Incubation of these polypeptides with 30,000 × g supernatant fractions from cowpea mesophyll protoplasts inoculated with complete virus or with separate bottom (B) components alone resulted in extensive processing, yielding polypeptides of 60, 58, 48, and 47 kilodaltons. Similar proteolytic activity was found associated with the in vitro translation products from the bottom-component RNA, demonstrating that the protease present in infected cells is encoded by B RNA. Using antisera raised against the separate capsid proteins VP23 and VP37, it was shown that the 60-kilodalton cleavage product is the precursor to both capsid proteins. Cleavage of nascent 95- and 105- kilodalton polypeptides by the in vivo protease demonstrated that this capsid protein precursor is located C terminally within both polypeptides and that the synthesis of these two overlapping polypeptides is the result of two initiation sites on middle-component RNA. In addition, a second virus-induced proteolytic activity, capable of releasing VP23 from the 95- and 105-kilodalton polypeptides, was detected in leaves of infected plants, but not in infected mesophyll protoplasts. A model for the expression of the middle-component RNA is presented.