Synthesis of functional mRNA in eukaryotes involves processing of precursor transcripts, including the addition of a poly(A) tail at the 3' end. A multiprotein complex recognizes a polyadenylation signal, generally the hexanucleotide AAUAAA in metazoans, to direct processing of the pre-mRNA. Based on sequence analysis of several cDNAs, we have previously suggested that the UAAA tetranucleotide (which may include the UAA translation stop codon) could be the polyadenylation signal in Trichomonas vaginalis, a parasitic protozoon that causes human trichomoniasis. This proposal is analyzed here with the aid of a transient-expression system of a reporter gene (cat flanked by T. vaginalis actin noncoding sequences). When cells were transfected with a plasmid bearing the original 3' untranslated region (UTR) sequence containing the UAAA motif, the resulting cat mRNA was polyadenylated similarly to the endogenous actin mRNA. Base changes in the UAAA sequence produced alterations to the polyadenylation site of the reporter mRNAs, while nucleotide substitutions at either side of UAAA did not. Furthermore, relocation of the UAAA motif redirected the processing and polyadenylation of the reporter mRNA. In addition, a pre-mRNA cleavage site for polyadenylation was defined. Interaction of T. vaginalis proteins with the UAAA motif was shown by electrophoretic mobility shift assays. Based on our findings, we provide evidence that in T. vaginalis the UAAA tetranucleotide has a role equivalent to that of the metazoan consensus AAUAAA polyadenylation signal.