The importance of sugar contacts for the sequences-pecific recognition that occurs during polyadenylation of mRNAs was investigated with chemically synthesized substrates containing 2′-OCH, groups at selected riboses. An RNA (5′-CUGCAAUAAACAAGU-UAA-3′) with 2′-OCH, ribose at each nucleotide except for the AAUAAA sequence and 3′-terminal adenosine was efficiently polyadenylated in vitro. Methylation of single riboses within AAUAAA inhibited both poly(A) addition and binding of the specificity factor, but the magnitude of inhibition varied greatly at different nucleotides. Nucleotides that showed sensitivity to base substitutions did not necessarily show sensitivity to ribose methylation, and vice versa. The data indicate that the specificity factor interacts with AAUAAA through RNA-protein contacts involving essential recognition of both sugars and bases at different nucleotide positions.