Eukaryotic post-transcriptional regulation is often specified by control elements within mRNA 3'- untranslated regions (3'-UTRs). In order to identify proteins that regulate specific mRNA decay rates in Saccharomyces cerevisae, we analyzed the role of five members of the Puf family present in the yeast genome (referred to as JSN1/PUF1, PUF2, PUF3, PUF4 and MPT5/PUF5). Yeast strains lacking all five Puf proteins showed differential expression of numerous yeast mRNAs. Examination of COX17 mRNA indicates that Puf3p specifically promotes decay of this mRNA by enhancing the rate of deadenylation and subsequent turnover. Puf3p also binds to the COX17 mRNA 3'-UTR in vitro. This indicates that the function of Puf proteins as specific regulators of mRNA deadenylation has been conserved throughout eukaryotes. In contrast to the case in Caenorhabditis elegans and Drosophila, yeast Puf3p does not affect translation of COX17 mRNA. These observations indicate that Puf proteins are likely to play a role in the control of transcript-specific rates of degradation in yeast by interacting directly with the mRNA turnover machinery.