We have shown previously that the decay of human bcl-2 mRNA is mediated by an adenine/uridine-rich element (ARE) located in the 3'-untranslated region. Here, we have utilized a non-radioactive cell-free mRNA decay system to investigate the biochemical and functional mechanisms regulating the ARE-dependent degradation of bcl-2 mRNA. Using RNA substrates, mutants, and competitors, we found that decay is specific and ARE-dependent, although maximized by the ARE-flanking regions. In unfractionated extracts from different cell types and in whole cells, the relative enzymatic activity was related to the amount of Bcl-2 protein expressed by the cells at steady state. The degradation activity was lost upon Bcl-2 depletion and was reconstituted by adding recombinant Bcl-2. Ineffective extracts from cells that constitutively do not express Bcl-2 acquire full degradation activity by adding recombinant Bcl-2 protein. We conclude that Bcl-2 is necessary to activate the degradation complex on the relevant RNA target.