Splicing of the human immunodeficiency virus type 1 (HIV-1) pre-mRNA must be inefficient to provide a pool of unspliced messages which encode viral proteins and serve as genomes for new virions. Negative cis-regulatory elements (exonic splicing silencers or ESSs) are necessary for HIV-1 splicing inhibition. We demonstrate that heterogeneous nuclear ribonucleoproteins (hnRNPs) of the A and B group are trans-acting factors required for the function of the tat exon 2 ESS. Depletion of hnRNP A/B proteins from HeLa cell nuclear extract activates splicing of tat exon 2 pre-mRNA substrate. Splicing inhibition is restored by addition of recombinant hnRNP A/B proteins to the depleted extract. A high-affinity hnRNP A1-binding sequence can substitute functionally for the ESS in tat exon 2. These results demonstrate that hnRNP A/B proteins are required for repression of HIV-1 splicing.