Members of the SR family of proteins, can collaborate with U1 snRNP in the recognition of 5' splice sites in pre-messenger RNAs. We have previously shown that purified U1 snRNP and ASF/SF2 form a ternary complex with pre-mRNA, which is dependent on a functional 5' splice site. In this manuscript we dissect the requirements for the formation of this complex. Sequences in the pre-mRNA, domains in ASF/SF2 and components of the U1 snRNP particle are shown to be required for complex formation. We had shown that sequences at the 5' splice site of PIP7. A are necessary and now we show these are sufficient for complex formation. Furthermore, we show that one functional RNA binding domain and the RS domain are both required for ASF/SF2 to participate in complex formation. The RNA binding domains were redundant in this assay, suggesting that either domain can interact with the pre-messenger RNA. Finally, our experiments show no function for the U1-specific A protein in complex formation, whereas a function for U1-specific C protein was strongly suggested. The study of the earliest interactions between pre-mRNA and splicing factors suggests a model for 5' splice site recognition.