The sequences that comprise a polyadenylation signal are varied. With the exception of the conserved hexanucleotide AAUAAA, other required sequence elements vary from site to site. This variability in sequence content may be indicative of different types or classes of polyadenylation signals. We have used an in vitro polyadenylation system to investigate the possibility that classes of poly(A) sites exist. Precursor RNAs from the SV40 late and adenovirus 2 L3 polyadenylation sites were examined for differences in: assembly into RNA-protein complexes; sequence requirements for complex assembly; and interactions with small nuclear ribonucleoproteins (snRNPs). Both SV40 late and L3 precursor RNA required an intact hexanucleotide and downstream sequence elements for complex formation. The stability of the complexes assembled using the two precursor RNAs was different. The L3 RNA complex was unstable in the presence of the anion poly(ACU); whereas the SV40 late complex or a chimeric L3/SV40 late complex were not. SV40 late and L3 precursor RNAs associated with the Sm protein determinant (common to U1, U2, U4/U6, U5 and U7 snRNPs) and a U1 snRNP-specific protein determinant early in the polyadenylation reaction. This association was reduced as the polyadenylation reaction progressed. Formation of the polyadenylation specific complexes was shown to require the small nuclear RNAs (snRNAs) U1, U2 and U4. The two polyadenylation precursor RNAs showed a different sensitivity to oligonucleotide directed RNase H cleavage of U RNAs. The SV40 late site was sensitive to cleavages of U1, U2 and U4 RNA. The L3 site showed sensitivity to only U4 cleavage. When the two polyadenylation signals were preceded by a functional intron with 5\sp′ and 3\sp′ splice sites, sensitivity to U RNA cleavages was altered. However even as chimeric polyadenylation splicing templates, the two sites exhibited different sensitivities to U4 cleavage in the region of U4 in which it hybridizes to U6 RNA. The observed differences in complex stability, and sensitivity to U RNA cleavage suggest that different classes of polyadenylation sites exist.