ARF GAP is a kind of important regulator of introcellular transport. Recently, a novel human gene has been found from a cDNA library of second trimester human fetal liver. The amino acid sequence encoded by the novel gene has 32% similarity to rat ARF1 GAP, was thus termed as ARFGAP3. Functional studies of the new gene were performed. The full-length cDNA of ARFGAP3 was amplified from the human total placenta RNA by RT-PCR technique, then subcloned into pGEM-T vector and sequenced. The RNA Master blot and multiple tissue Northern blot analysis were used to define the expression profile and the transcript size of ARFGAP3 in human tissues. It was shown that ARFGAP3 was strongly expressed in glands and testis and that ARFGAP3 mRNA existed as only one kind of transcript of 2.7 kb in various human tissues. Then, the expression and purification of the recombinant human ARFGAP3 (rhARFGAP3) were performed. It was demonstrated that rhARFGAP3 exhibited strong GTPase-activating protein (GAP) activity towards the recombinant ARF1 in vitro by an assay of a single round of GTP hydrolysis on recombinant ARF1, and that GAP activity of ARFGAP3 was stimulated by PIP2 and inhibited by PC.