During human immunodeficiency virus type 1 (HIV-1) virion assembly, cleavage of the Gag precursor by the viral protease results in the transient appearance of a nucleocapsid-p1-p6 intermediate product designated p15NC. Utilizing the p15NC precursor protein produced with an in vitro transcription-translation system or purified after expression in Escherichia coli, we have demonstrated that RNA is required for efficient cleavage of HIV p15NC. Gel mobility shift and nitrocellulose filter binding experiments indicate that purified p15NC protein specifically binds its corresponding mRNA with an estimated Kd of 1.5 nM. Binding was not affected by the presence or absence of zinc or EDTA. Moreover, mutagenesis of the cysteine residues within either of the two Cys-His arrays had no effect on RNA binding or on RNA-dependent cleavage by the viral protease. In contrast, decreased binding of RNA and diminished susceptibility to cleavage in vitro were observed with p15NC-containing mutations in one or more residues within the triplet of basic amino acids present in the region between the two zinc fingers. In addition, we found that 21- to 24-base DNA and RNA oligonucleotides of a particular sequence and secondary structure could substitute for p15 RNA in the enhancement of p15NC cleavage. Virus particles carrying a mutation in the triplet of NC basic residues (P3BE) show delayed cleavage of p15NC and a defect in core formation despite the eventual appearance of fully processed virion protein. These results define determinants of the p15NC-RNA interaction that lead to enhanced protease-mediated cleavage and demonstrate the importance of the triplet of basic residues in formation of the virus core.