The genome of all retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. We report the results of experiments designed to identify the 5' and 3' boundaries of the dimerization domain of the HIV-1 genome: (1) An HIV-1 RNA starting at nucleotide 252 or at other downstream positions (four tested) does not dimerize despite the inclusion of the whole of a previously proposed dimerization domain (nucleotides 295-401); (2) an RNA starting between nucleotides 242 and 249 (five positions tested) dimerizes to a variable extent depending on the starting position; (3) an RNA starting at nucleotide 233 or at other upstream positions (five tested) is fully or > 80% dimeric; (4) an RNA starting at nucleotide 1 but lacking the 233-251 or the 242-251 region is, respectively, fully monomeric or about 50% monomeric; (5) the 343-401 region contains two strings of G's (GGGGG367 and GGG384) that had been postulated to promote genome dimerization through the formation of guanine quartets. We have deleted the 379-401, 358-401, and 343-401 regions from otherwise dimeric RNAs without changing their ability to dimerize. We reach three conclusions: (1) a dimerization signal exists upstream of the major 5' splice donor (nucleotide 290); (2) the previously proposed downstream dimerization domain is insufficient to promote dimerization and has a 3' half that is not necessary to obtain fully dimeric RNAs; (3) the 5' boundary of the HIV-1 dimerization domain is located somewhere between nucleotides 233 and 242, and the 3' boundary is located no farther than at nucleotide 342, making it possible that the 5' and 3' boundaries of the HIV-1 dimerization domain are both located within the leader sequence. We speculate that the 248-270 or 233-285 region forms a hairpin that is the core dimerization domain of HIV-1 RNA.