The spliceosome deposits multiple proteins 20–24 nucleotides upstream of mRNA exon–exon junctions. This study shows that the spliceosome stably deposits several proteins on mRNAs, likely as a single complex of ~335 kDa. This complex protects 8 nucleotides of mRNA from complete RNase digestion at a conserved position 20–24 nucleotides upstream of exon-exon junctions. Splicing-dependent RNase protection of this region was observed in both HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei. Immunoprecipitations revealed that five components of the complex are the splicing-associated factors SRm160, DEK and RNPS1, the mRNA-associated shuttling protein Y14 and the mRNA export factor REF. Possible functions for this complex in nucleocytoplasmic transport of spliced mRNA, as well as the nonsense-mediated mRNA decay pathway, are discussed. The study demonstrates that pre-mRNA splicing stably alters mRNA structure at a conserved position, 20–24 nt upstream of mRNA exon–exon junctions, both in vitro and in vivo. At least five proteins become tightly associated with this region, probably as part of a single complex. The diverse attributes of these proteins may explain previously reported effects of pre-mRNA splicing on downstream mRNA metabolism. The -20/24 complex includes both pre-mRNA splicing and mRNA export factors. The -20/24 proteins remain stably bound to mRNA without apparent preference for a particular RNA sequence. The -20/24 proteins include both pre-mRNA splicing and mRNA export factors. The -20/24 complex has an apparent molecular mass of 335 kDa. The -20/24 complex suggests a role in NMD. The conserved position of the -20/24 complex suggests a role in NMD. The study also discusses the functional implications of the -20/24 complex for mRNA export and NMD.The spliceosome deposits multiple proteins 20–24 nucleotides upstream of mRNA exon–exon junctions. This study shows that the spliceosome stably deposits several proteins on mRNAs, likely as a single complex of ~335 kDa. This complex protects 8 nucleotides of mRNA from complete RNase digestion at a conserved position 20–24 nucleotides upstream of exon-exon junctions. Splicing-dependent RNase protection of this region was observed in both HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei. Immunoprecipitations revealed that five components of the complex are the splicing-associated factors SRm160, DEK and RNPS1, the mRNA-associated shuttling protein Y14 and the mRNA export factor REF. Possible functions for this complex in nucleocytoplasmic transport of spliced mRNA, as well as the nonsense-mediated mRNA decay pathway, are discussed. The study demonstrates that pre-mRNA splicing stably alters mRNA structure at a conserved position, 20–24 nt upstream of mRNA exon–exon junctions, both in vitro and in vivo. At least five proteins become tightly associated with this region, probably as part of a single complex. The diverse attributes of these proteins may explain previously reported effects of pre-mRNA splicing on downstream mRNA metabolism. The -20/24 complex includes both pre-mRNA splicing and mRNA export factors. The -20/24 proteins remain stably bound to mRNA without apparent preference for a particular RNA sequence. The -20/24 proteins include both pre-mRNA splicing and mRNA export factors. The -20/24 complex has an apparent molecular mass of 335 kDa. The -20/24 complex suggests a role in NMD. The conserved position of the -20/24 complex suggests a role in NMD. The study also discusses the functional implications of the -20/24 complex for mRNA export and NMD.