The human immunodeficiency virus type-1 matrix protein (HIV-1 MA) is a multifunctional structural protein synthesized as part of the Pr55 gag polyprotein. We have used in vitro genetic selection to identify an RNA consensus sequence that specifically interacts with MA (Kd = 5 × 10−7 M). This 13-nt MA binding consensus sequence bears a high degree of homology (77%) to a region (nt 1433–1446) within the POL open reading frame of the HIV-1 genome (consensus sequence from 38 HIV-1 strains). Chemical interference experiments identified the nucleotides within the MA binding consensus sequence involved in direct contact with MA. We further demonstrate that this RNA–protein interaction is mediated through a stretch of basic amino acids within MA. Mutations that disrupt the interaction between MA and its RNA binding site within the HIV-1 genome resulted in a measurable decrease in viral replication.