The possibility that internal initiation of translation is responsible for the synthesis of the middle component (M) RNA-encoded 95K protein of cowpea mosaic virus (CPMV) has been investigated by constructing plasmids in which the entire sequence of CPMV M RNA was cloned downstream of a chloramphenicol acetyltransferase gene. Expression of these plasmids in an animal cell expression system revealed that no synthesis of the proteins encoded by the downstream CPMV open reading frame takes place from RNA derived from these constructs under conditions where the internal ribosome entry site of foot-and-mouth disease virus is functional. The results indicate that internal initiation is not responsible for thc synthesis of the 95K protein in this system.