Base pairing between the 5′ end of U1 snRNA and the conserved 5′ splice site of pre-mRNA is important for commitment complex formation in vitro. However, the biochemical mechanisms by which pre-mRNA is initially recognized by the splicing machinery is not well understood. To evaluate the role of this base pairing interaction, we truncated U1 snRNA to eliminate the RNA–RNA interaction and surprisingly found that U1 snRNP can still form a nearly normal RNA–protein complex and maintain sequence specificity. We propose that some feature of U1 snRNP, perhaps one or more protein factors, is more important than the base pairing for initial 5′ splice site recognition. In addition, at least five sets of interactions contribute to complex formation or stability. Only one of these is base pairing between the 5′ splice site and the 5′ end of U1 snRNA, without which the U1 snRNP–pre-mRNA complex is less stable and has a somewhat altered conformation.