ARHGAP1, ARHGAP2, ARHGAP3, ARHGAP4, ARHGAP5, ARHGAP6, ARHGAP7 (DLC1), ARHGAP8, ARHGAP9, ARHGAP10, ARHGAP12, ARHGAP13 (SRGAP1), ARHGAP14 (SRGAP2), ARHGAP15, ARHGAP17 (RICH1), ARHGAP18, ARHGAP19, ARHGAP20, ARHGAP21, ARHGAP22, ARHGAP23, ARHGAP24, ARHGAP25, ARHGAP26, STARD13 (DLC2), HA-1, GMIP, PARG1, RACGAP1, PIK3R1, PIK3R2, and FNBP2 genes encode Rho/Rac/Cdc42-like GTPase activating (RhoGAP) proteins. Here, we characterized human ARHGAP27 gene by using bioinformatics. Complete coding sequence of ARHGAP27 isoform 1, encoding a full-length 889-aa protein, was determined by assembling exon 1 (nucleotide position 143440-144096 of AC091132.16) and most part of FLJ43547 cDNA (nucleotide position 69-3628 of AK125535.1). Complete coding sequence of ARHGAP27 isoform 2, encoding an N-terminally truncated 548-aa protein, was derived from FLJ43547 cDNA. ARHGAP27 isoform 1 consists of exons 1-17, while ARHGAP27 isoform 2 consists of exons 1B, and 2-17. ARHGAP27 gene encoded two isoforms due to alternative splicing of alternative promoter type. ARHGAP27 mRNA was expressed in germinal center B cell, spleen, chronic lymphocytic leukemia, pancreatic cancer, and lung cancer. LOC303583 (NM_ 198759.1) was the representative rat Arhgap27 cDNA. Human ARHGAP27 showed 84.3% total-amino-acid identity with rat Arhgap27, and 39.0% total-amino-acid identity with human ARHGAP12. ARHGAP27 and ARHGAP12 shared the common-domain structure, consisting of SH3, WW, PH, and RhoGAP domains. ARHGAP27 gene was located at human chromosome 17q21, while ARHGAP12 gene was located at human chromosome 10p11. ARHGAP family genes are cancer-associated genes, because their genetic alterations lead to carcinogenesis through the dysregulation of Rho/Rac/ Cdc42-like GTPases. This is the first report on identification and characterization of the ARHGAP27 gene.