The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5′ end of the intervening sequence joined to a site near the 3′ splice site. This lariat structure, which has been characterized for an adenovirus 2 major late transcript, has a branch point, with 2′-5′ and 3′-5′ phosphodiester bonds emanating from a single adenosine residue. The excised intervening sequence retains the branch site and terminates in a guanosine residue with a 3′ hydroxyl group. The phosphate group at the splice junction between the two exons originates from the 3′ splice site at the precursor.