The free energy yield from ATP hydrolysis is directly related to the [ATP]/ ([ADP] [Pi]) ratio within any given cell, and it has long been clear that cells can not maintain functionality when this ratio undergoes a significant change. This energetic challenge is most pronounced within the cells of excitable tissues with high and variable energy demands, cells such as cardiomyocytes, neurons, transport epithelia, and primitive free swimming spermatozoa. It is within these very cells that the creatine kinase (CK) system comes into play. CK catalyzes the reversible transfer of the ã-phosphoryl group from MgATP to the guanidine group of creatine, thereby maintaining ÄGATP by buffering the [ATP]/([ADP] [Pi]) ratio when there are temporal and spatial mismatches of ATP supply and ATP demand. Three distinct CK gene families exist – mitochondrial, cytoplasmic and flagellar – each targeted to different intracellular compartments. These genes appear to have evolved at the dawn of the radiation of multicellular animals. As part of on-going efforts to probe the evolutionary physiology in the CK gene family, the cDNAs for mitochondrial CKs from the protostome polychaete Chaetopterus variopedatus (CVMtCK) and chicken cardiac tissue (SarMtCK) have been cloned, inserted into an expression vector and recombinant protein expressed, purified and characterized. Recombinant CVMtCK was primarily octameric as was the well-characterized chicken SarMtCK. Using two independent methods (size-exclusion chromatography and dynamic light scattering, or DLS), studies of oligomerization dynamics showed that CVMtCK exhibited the same reversible transition between octamers and dimers as has been reported for MtCKs from higher organisms, and that these ancient octamers displayed the same dissociation and reassociation profile as that seen in the MtCKs from birds and man under various thermal and concentration regimes. However, the rate of change in both directions is much more rapid for CVMtCK. Interestingly, and perhaps importantly, when CVMtCK was converted to the transition state analog complex (TSAC) in the presence of NO3-, MgADP, and creatine, both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. To evaluate the potential structural correlates of the observed differences in octamer stability, a homology model was developed using the octameric crystal structures of SarMtCK and human ubiquitous UbiMtCK as templates. The resulting model was validated by a variety of on-line tools. Comparison of the structures showed some differences in the interactions occurring across the dimer - dimer interface which are likely to impact the stability of the octameric structure. In all structures, a key and absolutely conserved tryptophan residue is present in this interface. Site-directed mutagenesis procedures were employed to mutate this Trp residue to Cys, Phe, Leu and Tyr. In all cases, the specific activity was unaffected but the recombinant protein was dimeric; no octameric protein was detected using chromatography or DLS. The overall results of this effort show that octamer formation is a primitive character of MtCKs and that there has been some fine-tuning – in an evolutionary sense – of the nature of the interactions promoting and stabilizing the octameric state.