The chicken beta-tropomyosin pre-mRNA is spliced in a tissue-specific manner. Internal exons 6B and 6A are specifically used in skeletal muscle and non-skeletal muscle cells, respectively. Pre-mRNA secondary structure around exon 6B has been shown to be part of the mechanism that inhibits exon 6B to 7 splicing in HeLa nuclear extract. We analyse the influence of pre-mRNA folding on the different steps of spliceosome assembly under different conditions. At 3 mM MgCl2, conditions that favour RNA structure formation, the interactions of U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) with the pre-mRNA are all affected. The study of several mutants destabilising some proposed stem-loop structures shows that the in vitro splicing activation is correlated with an increased binding of snRNPs on pre-mRNA molecules. At 1 mM MgCl2, conditions that allow a partial relaxation of the inhibitory structure, U1 snRNP binding on exon 6B 5' splice site occurs very efficiently. Nonetheless, if this first step of spliceosome assembly is derepressed, U2, U4, U5 and U6 snRNP interaction processes remain inhibited. Altogether, these results suggest that the choice between exon 6A and 6B donor sites is a complex process not simply directed by a difference in the efficiency of interaction between U1 snRNP and alternative 5' splice sites.