Abstract: Biogenesis of cytochrome c oxidase is a complex process involving more than 30 known accessory proteins in yeast for the regulation of transcription and translation, membrane insertion and protein processing, cofactor insertion, and subunit assembly. Here, we focus on the process of cofactor insertion into subunit I of cytochrome c oxidase using the soil bacterium Paracoccus denitrificans as a model organism. The use of bacterial systems facilitates biogenesis studies, as the number of required assembly factors is reduced to a minimum. Both, co- and posttranslational cofactor insertion scenarios are discussed, and several approaches to shed light on this aspect of biogenesis are presented. CtaG, the Paracoccus homolog of yeast Cox11 which is involved in copper delivery to the Cu(B) center, has been purified and characterized spectroscopically. A previously unreported signal at 358 nm allows monitoring copper transfer from copper-loaded CtaG to an acceptor. Both CtaG and apo-subunit I were purified after expression in Escherichia coli to develop an in vitro copper transfer system, probing the posttranslational insertion hypothesis. To mimic a potential cotranslational insertion process, cell-free expression systems using E. coli and P. denitrificans extracts have been established. Expression of subunit I in the presence of the detergent Brij-35 produces high amounts of "solubilized" subunit I which can be purified in good yield. With this system it may be feasible to trap and purify assembly intermediates after adding free cofactors, purified assembly proteins, or P. denitrificans membranes.