Mitochondrial phenotype is complex and difficult to define at the level of individual cell types. Newer metabolic profiling methods provide information on dozens of metabolic pathways from a relatively small sample. This pilot study used “top-down” metabolic profiling to determine the spectrum of metabolites present in liver mitochondria. High resolution mass spectral analyses and multivariate statistical tests provided global metabolic information about mitochondria and showed that liver mitochondria possess a significant phenotype based on gender and genotype. The data also show that mitochondria contain a large number of unidentified chemicals.