RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AlMV) encode the replicase proteins P1 and P2, respectively. P1 expressed in transgenic plants (P1 plants) can be used in trans to support replication of AlMV RNAs 2 and 3, and P2 expressed in transgenic plants (P2 plants) can be used in trans to support replication of AlMV RNAs 1 and 3. Wild-type RNA 1 was able to coreplicate with RNAs 2 and 3 in P1 plants, but this ability was abolished by frameshifts or deletions in the P1 gene of RNA 1. Similarly, wild-type RNA 2 coreplicated with RNAs 1 and 3 in P2 plants, but frameshifts or deletions in the P2 gene of RNA 2 interfered with this replication. Apparently, the P1 and P2 genes are required in cis for the accumulation of RNAs 1 and 2, respectively. Point mutations in the GDD motif of the P2 gene in RNA 2 interfered with accumulation of RNA 2 in P2 plants, indicating that replication of RNA 2 is linked to its translation into a functional protein. Plants transformed with both the P1 and P2 genes (P12 plants) accumulate replicase activity that is able to replicate RNA 3 in trans. An analysis of the time course of the accumulation of RNAs 1, 2, and 3 in protoplasts of P12 plants supported the conclusion that translation and replication are tightly coupled for AlMV RNAs 1 and 2 but not for RNA 3.