Picornavirus-infected cells contain a double-stranded RNA (replicative form [RF] RNA), composed of viral genomic RNA hydrogen bonded to cRNA of similar nucleotide length. Mengovirus RF RNA reacted with a succinimide ester of biotin was shown by electron microscopy to bind avidin-coupled polymethacrylate spheres. These binding sites are taken to indicate the presence of VPg protein molecular by methods previously applied to poliovirus RF RNA (Richards et al., Proc. Natl. Acad. Sci. U.S.A. 76:676-680, 1979). One sphere was bound at or very near one terminus of linear RF molecules while a second sphere was bound at a site which was 1 to 4% of the genome length from the other terminus. Assignment of VPg positions was limited by the physical dimension (ca. 60-nm diameter) of the heavy metal-contrasted sphere observed by electron microscopy. A third site of sphere binding was detected at a lower frequency of occurrence at a site which was 10 to 20% of the genome length from one or the other terminus. Circular RF RNA molecules were also detected with two spheres attached at juxtaposed sites. The termini of the linear RF RNA were located within the circular structures by sphere attachment at sites which were very close to and obscured a nucleic acid projection which we have described to occur on circular mengovirus RF RNA (D. L. Robberson, M. V. Marshall, G. B. Thornton, and R. B. Arlinghaus, manuscript submitted for publication). CsCl gradient fractions of RNA reacted with avidin-coupled spheres were highly enriched in circular structures.