The late 19S RNAs of simian virus 40 (SV40) are functionally polycistronic, i.e., all encode both VP2 and VP3. The VP3-coding sequences are situated in the same reading frame as the VP2-coding sequences, within the carboxy-terminal two-thirds of the VP2-coding sequences. To test whether VP3 is produced by proteolytic processing of VP2, we introduced a variety of deletion and insertion mutations within the amino-terminal end of the VP2-coding sequences. Genetic and biochemical analysis of the proteins synthesized in cells transfected with these mutants indicated that VP2 and VP3 were synthesized independently of each other. A leaky scanning model for the synthesis of VP3 was tested by the insertion of a strong initiation signal (CCAACATGG) upstream of the VP3-coding sequences. When the signal was placed in the same reading frame as VP3, synthesis of VP3 was reduced by a factor of 10 to 20, whereas synthesis of the expected VP3-related fusion protein occurred at a rate similar to that observed for VP3 in cells transfected with wild-type SV40 DNA. Insertion of this strong initiation signal at the same site, but in a different reading frame, resulted in the synthesis of VP3 at one-third of the wild-type rate. Mutation of the VP2 initiator AUG resulted in a small but reproducible (1.6-fold) increase in VP3 accumulation. From these experiments we conclude that (i) VP3 is synthesized predominantly by independent initiation of translation via a leaky scanning mechanism, rather than by proteolytic processing of VP2 or direct internal initiation of translation; (ii) a strong initiation signal 5' of the VP3-coding sequences can significantly inhibit synthesis of VP3, but does not act as an absolute barrier to scanning ribosomes; (iii) approximately 70% of scanning ribosomes bypass the VP2 initiator AUG, which is present in a weak context (GGUCCAUGG), and initiate at the VP3 initiation signal located downstream; and (iv) reinitiation of translation appears to occur on the SV40 late 19S mRNAs at an efficiency of 25 to 50%.