The results of a previous study suggested that the poly(A) sequence in barley stripe mosaic virus (BSMV) RNA is intercalated between a 3'-terminal tyrosine-accepting structure and the 5'-terminal coding part of the BSMV genome. Here we show that poly(A)+ and poly(A)- fractions of BSMV RNA can be cleaved into two fragments specifically at the position of poly(A) or oligo(A) sequence with RNase H from Escherichia coli in the presence of oligo(dT)10. The shorter fragment (Sh) retains the ability of intact viral RNA to be aminoacylated, i.e., it represents the 3'-terminal part of BSMV RNA. Electrophoretic analysis of Sh-RNA reveals three closely positioned subspecies with an average length of about 210 nucleotides. The long 5'-terminal RNA fragment (L) produced by RNase H treatment has electrophoretic mobility similar to that of intact BSMV RNA, but displays neither amino acid-accepting ability nor infectivity. Nevertheless, L-RNA possesses the same messenger activity as the intact viral RNA and codes for the same pattern of polypeptides in rabbit reticulocyte lysate in vitro translation assays.