The mitochondrial pathway of apoptosis is initiated when proapoptotic BH3-only members of the Bcl-2 protein family are either upregulated or activated. The eight BH3-only proteins have distinct binding affinities for prosurvival Bcl-2 family members, and also for activating the pore-forming proteins Bak and Bax. However, due to the unfolded nature of BH3-only proteins, previous studies of their specificity have been limited to the use of BH3 peptides, in vitro translated proteins, and truncated recombinant proteins. To compare the ability of each BH3-only protein to activate Bak and Bax we developed stable recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk), and that of Bak and Bax. As expected, Bid chimeras were around 1000-fold more effective at activating Bak and Bax on mitochondria than the equivalent BH3 peptides. Notably, in contrast to a recent report, chimeras (and peptides) did not show a preference for activating Bak or Bak. Bid and Bim were the strongest activators, followed by Puma. Bik, Bmf and Hrk were weaker activators still. Neither Bad nor Noxa were activators. In addition, we found that the h0 position in the BH3 domain, recently found to be important for Bax activation, is also important for Bak activation.