Mutually exclusive splicing of exons 6A and 6B from the chicken -tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them. Splicing is the process by which introns from premessenger RNAs are removed in eukaryotes. Pre-mRNA splicing takes place within the spliceosome, which is a large, highly dynamic complex composed of four small ribonucleoprotein particles (snRNP 1 U1, U2, U4/U6, and U5) and multiple non-snRNP factors (1, 2). One of the most intriguing questions that remains in RNA splicing is how the 5Ј and 3Ј splice sites are selected and paired together within large RNA sequences (3). This question takes on particular importance in alternative splicing, where the selection of certain splice sites is modulated depending on the developmental stage, on tissue differentiation, or on metabolic changes of the cells (3). Numerous studies have demonstrated that regulatory sequences within the pre-mRNA that lie outside the splicing signals play a crucial role in controlling the choice of splicing sites in a given cellular context (reviewed in Refs. 4 and 5).Among these sequences are the splicing enhancers. These elements are found in a wide variety of metazoan pre-mRNAs, either within exons or introns. Purine-rich splicing enhancers (known as ESE) are a well characterized class of exonic splicing enhancers that mostly interact with specific subsets of SR proteins (reviewed in Refs. 6 and 7). SR proteins belong to a family of essential splicing factors that are highly conserved between Drosophila and mammals and that are involved in both constitutive and regulated splicing events (reviewed in Refs. 8 and 9). It has been proposed that the function of SR proteins is to stimulate the recognition of weak upstream 3Ј splice sites, by recruiting U2AF 65/35 , or to f...