Potential functional consequences of the differences in protein distribution between the mitochondria of the rat liver, heart, brain and kidney, as determined in the accompanying paper (26), were analyzed using a canonical metabolic pathway approach as well as a functional domain homology analysis. These data were inserted into the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway framework to give global and metabolic pathway specific information on the impact of the differential protein distribution on mitochondrial function. Custom pathway analysis was also performed using pathways limited to the mitochondrion. Using this approach, several well known functional differences between these mitochondrial populations were confirmed. These included GABA metabolism in the brain, urea synthesis in the liver, and the domination of oxidative phosphorylation in the heart. By comparing relative protein amounts of mitochondria across tissues, a greater understanding of functional emphasis is possible as well as the nuclear "programming" required to enhance a given function within the mitochondria. For proteins determined to be mitochondrial (26) and lacking a defined role functional domain BLAST analyses were performed. Several proteins associated with DNA structural modification and a novel CoA transferase were identified. A protein was also identified capable of catalyzing the first three steps of de novo pyrimidine synthesis. This analysis demonstrates that the distribution of nuclear encoded proteins significantly modifies the overall functional emphasis of the mitochondria to meet tissue specific needs. These studies demonstrate the existence of mitochondrial biochemical functions that at present are poorly defined.