We have examined the effects of base changes at the lariat branch site of a modified adenovirus major late precursor mRNA (pre-mRNA). Replacement of the A residue at the lariat attachment site with a G residue was studied. Incubation of this altered pre-mRNA with nuclear extracts of HeLa cells yielded less spliced mRNA (10-fold) than similar reactions with the wild type pre-mRNA. The intron lariat formed during the reaction with the mutant transcript contained the predominant branch (2'-5' phosphodiester linkage) to an upstream A residue. In contrast, the intron/exon 2 lariat contained the predominant branch to the substituted G residue. These results indicated that detectable spliced RNA was formed when the lariat was attached at the A residue but not when the lariat was attached to the substituted G residue. A second mutation was introduced into the transcript by substituting an additional G residue at the alternative A branch site. When transcript derived from this plasmid was incubated with nuclear extract, cleavage occurred at the 5' splice site, and an intron/exon 2 lariat was produced, but spliced RNA was not detected. T1 RNase digestion and primer extension analyses of this intron/exon 2 lariat revealed that all of the lariat formed on the G residue at the normal attachment site.