Antisense RNA-mediated regulation in bacterial systems is related to the kinetics of RNA-RNA annealing in vitro. Here, we investigated the secondary structure of αY69, an effective HIV-directed antisense RNA in human cells. Purified RNA preparations contain a single conformer. The global structure was identified by a cleavage experiment under native conditions using a short complementary oligonucleotide and RNase H. Structural analyses indicate a three-domain structure of αY69 consisting of two stem-loop elements connected by a seven-nucleotide single-stranded hinge region. Kinetic data suggest that the formation of base pairs between a CGC triplet of αY69 and its target RNA is essential for fast annealing. The complementary sequence stretch of the target folds into a high-energy secondary structure. The relationship between modifications in structural elements of αY69 and the annealing kinetics suggested that rate-limiting steps of the annealing involve a single site of αY69 and do not involve its 5' or 3'-end. Further, the data indicate that both initial base-specific interactions and duplex formation are dependent on the CGC triplet of the central region of αY69. This mechanism represents a specific and efficient way of RNA-RNA annealing that is initiated by the interaction of unstructured RNA regions.