The 5' end of avian sarcoma and leukosis virus RNA near the primer binding site forms two RNA secondary structure, U5-inverted repeat (U5-IR) and U5-leader stems, which are required for efficient initiation of reverse transcription. Lying between these two secondary structures is a 7-base sequence that can anneal to the T-psi-C loop of the tRNA(Trp) primer. Base substitutions in U5 RNA which disrupt this potential interaction result in a defect in the initiation of reverse transcription both in vivo and in vitro. The defect can be complemented in vitro by base substitutions in the primer. The U5 RNA-T-psi-C interaction is also dependent upon the presence of both the U5-IR and the U5-leader structures. These RNA secondary structures and primer interactions are conserved in other type C and D retroviruses, suggesting that there is a common mechanism for the initiation of reverse transcription in all of these retroviruses.