The Obg subfamily protein is one of the P-loop small G proteins and is highly conserved in many organisms from bacteria to human. Two obg genes, obgH1 and obgH2, exist in the human genome. Both ObgH1 and ObgH2 showed similar GTPase activities (0.014 +/- 0.005 and 0.010 +/- 0.002/min for ObgH1 and ObgH2, respectively) to those of the bacterial Obg proteins and complemented the Obg function in Escherichia coli ribosome maturation, suggesting that the functions of Obg proteins are well conserved through evolution. Immunofluorescence microscopy of HeLa cells revealed that ObgH1 localizes in mitochondria, and ObgH2 in the dense fibrillar compartment region of the nucleolus. Knock-down of ObgH1 by RNAi induced mitochondria elongation, whereas knock-down of ObgH2 resulted in the disorganization of the nucleolar architecture. In conclusion, the two human Obg proteins have similar enzymatic activities that can complement bacterial Obg function, but show different cellular function(s) with different intracellular localizations.