The four encapsidated RNAs of brome mosaic virus (BMV; B1, B2, B3, and B4) contain a highly conserved 3' 200-nucleotide (nt) region encompassing the tRNA-like structure (TLS) which is required for packaging in vitro (Y. G. Choi, T. W. Dreher, and A. L. N. Rao, Proc. Natl. Acad. Sci. USA 99655-660, 2002). To validate these observations in vivo, we performed packaging assays using Agrobacterium-mediated transient expression of RNAs and coat protein (CP) (P. Annamalai and A. L. N. Rao, Virology 33896-111, 2005). Coexpression of TLS-less constructs of B1 or B2 or B3 and CP mRNAs in Nicotiana benthamiana leaves resulted in packaging of TLS-less B1 and B2 but not B3, suggesting that packaging of B3 requires the TLS in cis. This conjecture was confirmed by the efficient packaging of a B3 chimera in which the viral TLS was replaced with a cellular tRNA(Tyr). When N. benthamiana leaves were infiltrated with a mixture of transformants containing wild-type B1 (wtB1) plus wtB2 plus a TLS-less B3 (wtB1+wtB2+TLS-lessB3), the 3' end of progeny B3 was restored by heterologous recombination with that of either B1 or B2. This intrinsic cis-requirement of TLS in promoting B3 packaging was further confirmed when a mixture containing agrotransformants of TLS-less B1+B2+B3 was supplemented with either wtB4 or a 3' 200-nt or 3' 336-nt untranslated region (UTR) of B3. Northern blot analysis followed by sequencing of B3 progeny revealed that replication of TLS-less B3, but not TLS-less B1 or B2, was fully restored due to recombination with TLS from transiently expressed wtB4 or the B3 3' UTR. Collectively, these observations suggested that the requirement of a cis-acting TLS is distinct for B3 compared with B1 or B2.