Invadopodia are cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing a BAR domain, is a substrate of Src. ASAP1 is required for the assembly of invadopodia and podosomes, which are Src-induced structures related to invadopodia in NIH 3T3 fibroblasts. The BAR domain of ASAP1 is required for the assembly of podosomes. Using two-hybrid screening, we have identified GEFH1, a guanine nucleotide exchange factor for RhoA, as a binding partner of the BAR domain of ASAP]. We validated the interaction of endogenous GEFH1 with ASAP1 by immunoprecipitation, and found GEFH1 colocalized with ASAP1 in podosomes. The overexpression of GEFH1 inhibited podosome assembly and ASAP1 catalytic activity as a GAP. A mutant of GEFH1 lacking the domain that binds to the BAR domain of ASAP] was less effective. Reduced expression of GEFH1, achieved with siRNA treatment, did not affect matrix degradation by podosomes but increased the rate of podosome assembly. Based on these results, we conclude that GEFH1 is a negative regulator of podosomes. Published by Elsevier Inc.