The mammalian adaptor protein Alix participates in multiple cellular processes. Since mouse Alix cDNA detects two distinct transcripts of ∼3.5 and ∼7.0 kb in various mouse tissues, it is possible that there exist isoforms of Alix protein that perform varied biological functions. In this study, we first demonstrate that four different anti-Alix monoclonal antibodies immunoblot the single Alix protein in nine different mouse tissues. We then show that the two transcripts of 3.2 and 6.4 kb are widely expressed in various human tissues and cell lines. These two transcripts are generated from the same Alix gene localizing at 3p22.3 via alternative polyadenylation, thus containing an identical open reading frame. However, the 3.2-kb transcript is much more active in translation than the 6.4-kb transcript in a randomly selected cell line. These results eliminate the possibility that the two transcript variants encode different isoforms of Alix protein and suggest that alternative polyadenylation is one of the mechanisms controlling Alix protein expression.