The secondary structure of the adenovirus-2 L4 polyadenylation domain was established using enzymatic attacks and chemical modifications. This domain is characterized by the co-existence of several variants including stem-loop structures of low stability such that its secondary structure is clearly distinct from that of the upstream sequence which harbors large stable hairpin structures. The L4 polyadenylation site is located in a free single-stranded region and the AAUAAA signal is exposed in the 14 nucleotide loop of a hairpin structure with a six-base-pair stem. The 5' arm of this stem includes six nucleotides of a previously identified upstream efficiency element (USEa), whose deletion or substitution reduces cleavage-polyadenylation efficiency by 50%. Several evidences suggest that a signal hairpin structure may be important for polyadenylation. First, the AAUAAA signal is well exposed to enzymes and reagents in its two co-existing variants, suggesting that it is also accessible to cleavage-polyadenylation factors. Second, the disruption of the stem induces a 50% decrease of cleavage efficiency in vitro. Third, the reduction of the size of the loop to five nucleotides reduces cleavage efficiency by 90%. Fourth, polyadenylation domains of other origins can be folded into hairpin structures harboring the AAUAAA signal in their loop. We propose that one of the functions of an upstream efficiency element could be to participate in the presentation of the signal to trans-acting factors.