ARL5 is it member of ARLs, which is widespread in high eukaryotes and homologous between species, But no structure or biological function of this member is reported. We expressed, purified. and resolved the structure of human ARL5 with bound GDP3'P at 2.0 angstrom resolution. A comparison with the known structures of ARFs shows that besides the typical features of ARFs, human ARL5 has specific features of its own. Bacterially expressed human ARLS contains bound 61311311 which is seldom seen in other structures. The hydrophobic tail of the introduced detergent Triton X-305 binds at the possible myristoylation site of Gly(2), simulating the myristoylated state of N-terminal amphipathic helix in vivo. The structural features of the nucleotide binding motifs and the switch regions prove that ARL5 will undergo the typical GDP/GTP structural cycle as other members of ARLs, which is the basis of their biological functions. (c) 2005 Elsevier Inc. All rights reserved.