RNase H and synthetic DNA oligonucleotides were used to analyze the ribonucleoprotein (RNP) structure of the yeast spliceosome and to assay the pre-mRNA sequence requirements for step 1 of splicing. The data suggest that tight, stable contacts between the pre-mRNA and the spliceosome may be limited to the 5' splice site and branch point regions of the intron. A 30 nucleotide segment 3' of the branch point was found to be necessary for spliceosome maturation and essential for step 1 of splicing. Somewhat surprisingly, the 3' splice site was sensitive to nuclease digestion and completely dispensable for step 1 of splicing.