Abstract Biochemical components (splicing factors) interact with specific intron regions during pre-mRNA splicing in vitro. The pre-mRNA specifically associates with factors at both the branch point and the 5′ splice site and these RNA-factor interactions are maintained in the intron-containing RNA processing products. The first detectable event, the ATP-dependent association of a factor (or factors) with the branch point, is mediated by at least one factor containing an essential nucleic acid component. Mutant RNA substrates that lack either the 5′ splice site or the vast majority of exon sequences can still associate with the branch point binding factor(s). However, this branch point-factor interaction does not occur with a mutant RNA substrate that contains the branch point but that lacks the 3′ splice site consensus sequence. These results suggest that selection of the 3′ splice site accompanied by the association of a factor with the branch point may be the initial step in mammalian pre-mRNA splicing.