Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudo exons flanked by pseudo splice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudo exon splicing, we have systematically altered known splicing signals as well as immediately adjacent flanking sequences of an arbitrarily chosen pseudo exon from intron 1 of the human hprt gene. The substitution of a 5' splice site that perfectly matches the 5’ consensus combined with mutation to match the CAGG of the 3' consensus failed to get this model pseudo exon included as the central exon in a dhfr minigene context. Provision of a real 3' splice site and a consensus 5' splice and the removal of an upstream inhibitory sequence was necessary and sufficient to confer splicing on the pseudo exon. This activated context also supported the splicing of a second pseudo exon sequence containing no apparent enhancer. Thus both the 5’ splice site sequence and the polypyrimidine tract of the pseudo exon are defective despite their good agreement with the consensus. On the other hand, the pseudo exon body did not exert a negative influence on splicing. The introduction into the pseudo exon of a sequence selected for binding to ASF/SF2 or its substitution with b-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition, and suggest that intrinsically defective splice sites and negative elements may play important roles in distinguishing the real from the vast number of false splicing signals.