Abstract RNA 3 of alfalfa mosaic virus (AlMV) encodes the movement protein P3 and the viral coat protein (CP) which is translated from the subgenomic RNA 4. RNA 3 is able to replicate in tobacco plants transformed with the AlMV replicase genes P1 and P2 (P12 plants). Frameshifts or deletions in the P3 gene have little effect on RNA 3 accumulation in P12 protoplasts whereas such mutations in the CP gene result in a 100-fold reduction of plus-strand RNA 3 accumulation. When P12 protoplasts were inoculated with a mixture of a RNA 3 mutant with a deletion in the P3 gene and a mutant with a deletion in the CP gene, CP expressed by the P3 mutant was unable to upregulate plus-strand RNA accumulation of the CP mutant. However, when a wild-type CP gene and subgenomic promoter were inserted in a RNA 3 mutant with a defective CP gene, the mutant accumulated at wild-type levels. It is concluded that the function of CP in plus-strand RNA 3 accumulation acts in cisand cannot be complemented in trans.In P12 plants, P3 and CP mutants were able to complement each other at low and variable levels. This complementation in plants appeared to be correlated with the occurrence of recombination to wild-type RNA 3.