The function of human immunodeficiency virus nef gene product has been much debated but the precise activity of this protein in the HIV replication cycle remains unknown. HIV-1 Nef was obtained as a fusion protein with maltose binding protein (MBF), purified by amylose column chromatography and separated from MBP by cleavage with factor Xa. Purified HIV-1 Nef protein, but not the fusion protein MBP-Nef, binds to RNA in vitro as tested by three different assays, radioactive or non-radioactive. North-western analysis, UV cross-linking or band-shift analysis. This activity was lost in a deletion mutant lacking 22 amino acids from the amino terminus of HIV-1 Nef, while a deletion of 44 residues from the carboxy terminus of the protein does not impair the RNA binding activity. Moreover, a single amino acid replacement, Arg to Gly at position 22 produces a Nef variant deficient in its ability to interact with RNA. Different Nef proteins from HIV-1, HIV-2 or SIV were fused to MBP and cleaved with factor Xa. The different Nef proteins were all endowed with RNA-binding capacity. Sequence similarities between several RNA binding proteins, including picornavirus 2C and different Nef proteins are observed. The function of Nef during the HIV replication cycle is discussed on the basis of the present findings.