The formation of mature mRNAs in vertebrates involves the cleavage and polyadenylation of the pre-mRNA, 10-30 nt downstream of an AAUAAA or AUUAAA signal sequence. The extensive cDNA data now available shows that these hexamers are not strictly conserved. In order to identify variant polyadenylation signals on a large scale, we compared over 8700 human 3' untranslated sequences to 157,775 polyadenylated expressed sequence tags (ESTs), used as markers of actual mRNA 3' ends. About 5600 EST-supported putative mRNA 3' ends were collected and analyzed for significant hexameric sequences. Known polyadenylation signals were found in only 73% of the 3' fragments. Ten single-base variants of the AAUAAA sequence were identified with a highly significant occurrence rate, potentially representing 14.9% of the actual polyadenylation signals. Of the mRNAs, 28.6% displayed two or more polyadenylation sites. In these mRNAs, the poly(A) sites proximal to the coding sequence tend to use variant signals more often, while the 3'-most site tends to use a canonical signal. The average number of ESTs associated with each signal type suggests that variant signals (including the common AUUAAA) are processed less efficiently than the canonical signal and could therefore be selected for regulatory purposes. However, the position of the site in the untranslated region may also play a role in polyadenylation rate.