A biologically important human gene, cyclooxygenase-2 (COX-2), has been proposed to be regulated at many levels. While COX-1 is constitutively expressed in cells, COX-2 is inducible and is upregulated in response to many signals. Since increased transcriptional activity accounts for only part of the upregulation of COX-2, we chose to explore other RNA processing mechanisms in the regulation of this gene. We performed a comprehensive bioinformatics survey, the first of its kind known for human COX-2, which revealed that the human COX-2 gene has alternative polyadenylation (proximal and distal sites) and suggested that use of the alternative polyadenylation signals has tissue specificity. We experimentally established this in HepG2 and HT29 cells. We used an in vivo polyadenylation assay to examine the relative strength of the COX-2 proximal and distal polyadenylation signals, and have shown that the proximal polyadenylation signal is much weaker than the distal one. The efficiency of utilization of many suboptimal mammalian polyadenylation signals is affected by sequence elements located upstream of the AAUAAA, known as upstream efficiency elements (USEs). Here, we used in vivo polyadenylation assays in multiple cell lines to demonstrate that the COX-2 proximal polyadenylation signal contains USEs, mutation of the USEs substantially decreased usage of the proximal signal, and that USE spacing relative to the polyadenylation signal was significant. In addition, mutation of the COX-2 proximal polyadenylation signal to a more optimal sequence enhanced polyadenylation efficiency 3.5-fold. Our data suggest for the first time that alternative polyadenylation of COX-2 is an important post-transcriptional regulatory event.