Introns are excised from full-length transcripts (pre-messenger RNAs) of eukaryotic genes in two steps. First, the pre-mRNA is cleaved at the 5' splice site and a branched (lariat) intermediate is formed. Then, cleavage at the 3' splice site and ligation of the two exons leads to the release of the lariat intron. The intron sequence which accepts the 5' end to form the lariat branch is strictly conserved in yeast, but shows more variation in eukaryotes. To investigate the requirements for branch formation in eukaryotes further, we have studied in vitro splicing of a rabbit globin gene intron with mutations of the normal branch-accepting adenosine nucleotide. We conclude that all four nucleotides can serve as branch acceptors, but that A and C are preferred to G and U in lariat formation. Mutation of the normal A to G or U can lead to an A residue one nucleotide upstream of the normal branch site being used instead. Only branches to A or C participate efficiently in the second splicing step.