Three sequences in the vicinity of poly (A) addition sites are conserved among vertebrate mRNAs. We analyze the effects of single base changes in each position of AAUAAA and in the nucleotlde to which poly (A) is added on 3′ end formation in vitro. All 18 possible single base changes of the AAUAAA sequence greatly reduce addition of poly (A) to RNAs that end at the poly (A) addition site, and prevent cleavage of RNAs that extend beyond. The magnitude of reduction varies greatly with the position changed and the base introduced. For any given mutation, cleavage and polyadenylation are reduced to similar extents, strongly suggesting that the same factor interacts with AAUAAA in both reactions. Mutations at and near the conserved adenosine to which poly (A) Is added disturb the accuracy, but not the efficiency, of 3′ end formation. For example, point mutations at the conserved adenosine shift the 3′ end of the most abundant 5′ half-molecule downstream by a single nucleotide. The mechanism by which these mutations might exert their effects on the precision of 3′ end formation are discussed.