The binding of five barley proteins (Mr: 37, 36, 35, 34, and 30 kDa) to the ICR2 motif present at the 5' end of brome mosaic virus (BMV) RNA-2 was identified using UV cross-linking. Evidence that the interaction is specific included the observation that these proteins did not recognize a similar-size RNA fragment transcribed from a nonviral (beta-glucuronidase) gene, nor did they bind to the 3' end of the plus strand of RNA-3. Replication-defective BMV RNA-2 mutants bearing substitution mutations at nucleotides 9 and 10 of the ICR2 motif were used to show that these positions were involved in the interaction of the five barley proteins with BMV RNA-2. Surprisingly, the profile of barley proteins interacting with the 3' end of the minus strand of RNA-2 was similar to that seen for the 5' end of the plus strand. Further, the profile of proteins binding to minus-sense probes bearing substitution mutations in the ICR2 region differed from that found for the wild-type sequence. These findings support the concept that host proteins are involved in genome replication and that their ability to interact with both plus and minus strands of the viral RNA is probably involved with the initiation of plus-strand synthesis.