The coat protein of alfalfa mosaic virus has both structural and regulating functions. The latter is evident from the fact that the genomic RNAs of the virus, although they are of messenger polarity, cannot start an infection cycle in the absence of cost protein. The reason could be that the coat protein is needed for viral RNA synthesis. Indeed, the coat protein has been found in tight association with the viral RNA polymerase (R. Quadt et al., 1991, Virology 182, 309-315). To investigate the role of the coat protein, if any, in viral RNA synthesis, we have isolated that viral RNA polymerase (RNA-dependent RNA polymerase, RdRp) from mock-inoculated tobacco plants transformed with cDNAs 1 and 2, known as P12 plants (P. E. M. Taschner et al., 1991, Virology 181, 687-693), which express the nonstructural proteins P1 and P2. Such an enzyme (called M-RdRp) will contain the viral subunits P1 and P2 but not the coat protein. As a comparison we also isolated the RdRp from virion-inoculated P12 plants (C-RdRp). This enzyme will contain the coat protein. We found that both M-RdRp and C-RdRp could synthesize minus RNA, showing that coat protein is not needed for minus-strand synthesis. In contrast, minus-strand synthesis by both enzymes was inhibited by coat protein. Plus-strand synthesis was unaffected by coat protein in the case of C-RdRp, but strongly stimulated by coat protein in the case of M-RdRp. These data might explain why infected cells, which do not produce coat protein, display a very low accumulation of viral plus-strand RNA. They also give a possible explanation for the noninfectious character of the genomes of alfalfa mosaic virus and ilarviruses in the absence of coat protein. The fact that an active enzyme could be isolated from the same membrane fraction in infected and noninfected P12 plants shows that coat protein is not needed for assembly and targeting of the viral RNA polymerase.