We have previously demonstrated that an exon splicing silencer (ESS) is present within human immunodeficiency virus type 1 (HIV-1)tat exon 2. This 20 nucleotide (nt) RNA element acts selectively to inhibit splicing at the upstream 3'splice site (3'ss #3) flanking this exon. In this report, we have used in vitro splicing of mutated RNA substrates to determine the sequences necessary and sufficient for the activity of the ESS. The activity of the ESS within tat exon 2 maps to a 10 nt core sequence CUAGACUAGA. This core sequence was sufficient to inhibit splicing when inserted downstream from the 3'ss of the heterologous Rous sarcoma virus src gene. Mutagenesis of the interspersed purines in the polypyrimidine tract of the tat exon 2 3'ss to pyrimidines resulted in a significant increase in splicing efficiency indicating that 3'ss#3 is suboptimal. The ESS acts to inhibit splicing at the optimized 3'splice sites of both the HIV-1 tat and RSV src constructs but with a reduced efficiency compared to its effect on suboptimal 3'splice sites. The results indicate that both the ESS and a suboptimal 3'splice site act together to control splicing at the 3'splice site flanking at exon 2.