Antisense RNAs can specifically and efficiently inhibit replication of the human immunodeficiency virus type 1 (HIV-1). One of the most effective viral target regions covers the first coding exons of the viral regulator genes tat and rev. Large parts of the corresponding antisense RNAs of several hundred nucleotides in length could be removed without loss of inhibitory efficiency. The smallest antisense RNA tested (αY69, 69 nucleotides in length) showed an enhanced inhibitory effect in human cells. Its secondary structure was analysed experimentally and was found to fold into a Y-shaped structure composed of two linked stem/loop structures with loop sizes of 5 and 10 nucleotides, respectively. A similar structural element was found to be formed by its complementary HIV-1-derived 645 nt long target RNA (SR6). Kinetic analyses of double strand formation between αY69 and SR6 led to a second order rate constant of k = 2.9 × 10 4 M -1 s −1 at 37°C and physiological ionic strength. The large loop of αY69 plays a crucial role in the hybridization process in vitro as shown by kinetic analyses of a set of mutants derived from αY69. Base exchanges in loop regions resulted in an up to 10-fold lower association rate constant while exchanges in stem regions of αY69 had no effect in vitro. We discuss a model for the pairing mechanism in vitro in which not only the first and reversible interactions (termed "kissing" in well documented cases) but also subsequent steps leading to complete RXA duplex formation make use of the large loops located at complementary positions on both RNA strands.