RhoGAP family proteins, encoded by ARHGAP family genes, are negative regulators of Rho family GTPases, which are implicated in actin remodeling, cell polarity control, and cell migration. Based on the homology with ARHGAP22, we identified and characterized two novel ARHGAP family genes, ARHGAP24 and ARHGAP25. FLJ33877 cDNA (AK091196.1) and aberrant DKFZp564- B1162 cDNA (NM_031305.1) were derived from human ARHGAP24 gene. Two isoforms of KIAA0053 type (D29642.1) and BM927439 type were derived from human ARHGAP25 gene due to alternative splicing (alternative promoter). Mouse 0610025G21 (NM_029270.1) and A130039I20 (AK037710.1) were representative cDNAs derived from mouse Arhgap24 and Arhgap25 genes, respectively. Exon-intron structure of ARHGAP25 gene at human chromosome 2p13 was slightly divergent from that of ARHGAP22 and ARHGAP24 genes. MGC35285, MAPK8 and C10orf64 genes linked to ARHGAP22 gene were paralogs of PTPN13, MAPK10 and WDFY3 genes linked to ARHGAP24 gene, respectively. MGC35285-ARHGAP22-MAPK8-C10orf64 locus at human chromosome 10q11 and the WDFY3-ARHGAP24-MAPK10-PTPN13 locus at human chromosome 4q21 were paralogous regions (paralogons) within the human genome. Human ARHGAP24 showed 91.8% and 48.6% total-amino-acid identity with mouse Arhgap24 and human ARHGAP22, respectively. Human ARHGAP25 showed 86.1% and 40.8% total-amino-acid identity with mouse Arhgap25 and human ARHGAP22, respectively. ARHGAP22, ARHGAP24 and ARHGAP25 were found to constitute the RhoGAP subfamily featured by Pleckstrin homology (PH) domain and C-terminal Coiled-coil domain. This is the first report on identification and characterization of the ARHGAP24 and ARHGAP25 genes.