We used several related pre-mRNA substrates consisting of two introns and three exons to study effects of exon sequences on in vitro splicing. By varying the sequence of the internal exon and measuring the frequency of its skipping we confirmed that 26-nucleotide exon element naturally existing in beta-globin gene and previously analysed in vivo, has a strong stimulatory effect on splicing. Sequence analysis of this element suggests that it belongs to a family of purine-rich splicing elements found in exons of several alternatively spliced pre-mRNAs. The 26-nucleotide element can efficiently function in enhancing inclusion of internal exons regardless of their size and sequence composition, suggesting that it plays a role of a general exon recognition element. The purine-rich element is dispensable in exons flanked by strong splice sites, which promote efficient inclusion of otherwise poorly recognized exons. A row of six cytidines inserted into the internal exon (GC2 mutation) initially considered to stimulate exon inclusion to a similar extent as the purine-rich element (Dominski & Kole, 1994, J. Biol. Chem. 269, 23590-23596), appears not to affect splice site selection in vitro, and in vivo it is likely to act by stabilizing mRNA that includes the internal exon against rapid cytoplasmic degradation.