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To facilitate profiling mitochondrial transcriptomes, we developed a third-generation human mitochondria-focused cDNA microarray (hMitChip3) and its bioinformatic tools. hMitChip3 consists of the 37 mitochondrial DNA-encoded genes, 1098 nuclear DNA-encoded and mitochondria-related genes, and 225 controls, each in triplicate. The bioinformatic tools included data analysis procedures and customized database for interpretation of results. The database associated 645 molecular functions with 946 hMitChip3 genes, 612 biological processes with 930 genes, 172 cellular components with 869 genes, 107 biological chemistry pathways with 476 genes, 23 reactome events with 227 genes, 320 genetic disorders with 237 genes, and 87 drugs targets with 55 genes. To test these tools, hMitChip3 was used to compare expression profiles between human melanoma cell lines UACC903 (rapidly dividing) and UACC903(+6) (slowly dividing). Our results demonstrated internal geneset consistency (correlation R ≥ 0.980 ± 0.005) and interexperimental reproducibility (R ≥ 0.931 ± 0.013). Expression patterns of 16 genes, involved in DNA, RNA, or protein biosyntheses in mitochondrial and other organelles, were consistent with the proliferation rates of both cell lines, and the patterns of 6 tested genes were verified by quantitative reverse transcription PCR (RT-PCR). Thus, hMitChip3 and its bioinformatics software provide an integrated tool for profiling mitochondria-focused gene expression.