Multimerization of the human immunodeficiency virus type 1 (HIV-1) Rev protein is believed to be critical to its biological activity. However, the precise protein sequence requirements for Rev multimerization in vivo, and whether multimerization is facilitated by specific RNA binding or vice versa, has remained controversial. In this report, we describe a sensitive in vivo assay for the multimerization of HIV-1 Rev on its cognate RRE primary RNA binding site. Using this assay, we demonstrate that an intact Rev arginine-rich domain, while critical to specific RNA binding, is dispensable for multimerization on the RRE. Mutations introduced into Rev sequences that flank this basic domain produce a partial multimerization phenotype in vivo even though these mutations are known to block Rev multimerization in vitro. Similarly, mutations introduced into the leucine-rich activation domain of Rev, which appear to have no effect on in vitro multimerization, also markedly inhibit multimerization of Rev on the RRE in vivo. Overall, these data appear consistent with the hypothesis that in vivo formation of the multimeric Rev:RRE ribonucleoprotein complex is facilitated by both the RRE RNA substrate and, as first proposed by Bogerd and Greene U. Virol. 67, 2496-2502, 1993), by bridging by a cellular cofactor for Rev that likely interacts with multiple Rev activation domains.