Polyadenylation (poly(A)) of eukaryotic mRNA is a critical step for gene expression. In plants, poly(A) signals leading to the formation of polyadenosine tails after mRNAs include the far upstream elements, the AAUAAA-like signals, and the mRNA cleavage sites for poly(A). Multiple AAUAAA signals leading to alternative polyadenosine formation have been found in many genes, but the effects of each AAUAAA signal on gene expression remain to be uncovered. A DNA fragment, whose transcript contains two canonical AAUAAA signals from the 3'-untranslation region of endochitinase gene of tobacco (Nicotiana tabacum L. cv. W38), was mutated and constructed into the downstream of beta-glucuronidase (GUS) coding region. Transient expression of GUS gene from these constructs indicated that the distal AAUAAA signal from the stop codon was more important than the proximal one in stimulating gene expression. Also, the sequence rather than the distance between the stop codon and the AAUAAA signal region was critical for gene expression. Transgenic tobaccos with these constructs were also generated, and the position of the polyadenosine tail formation in this region was mapped. Results revealed that both AAUAAA signals were functional, and that polyadenosine tails of most transcripts were directed by the distal AAUAAA signal. Finally, the RNA stabilities of these variants in transgenic plants were measured. RNAs from the variants with the functional distal AAUAAA signal were more stable than those with the functional proximal one only. The possible secondary structure in this poly(A) signal region was predicted and discussed.