Rho family GTPases are important regulators of neuronal morphology, but the proteins directly controlling their activity in neurons are still poorly defined. We report the identification of myr 7, a novel unconventional myosin IX-RhoGAP expressed in rat brain. Myr 7 is a multidomain protein related to myr 5, the first class IX myosin to be characterized. It exhibits a myosin head domain with an N-terminal extension and a large insertion at loop 2, an actin contact site and regulator of myosin ATPase rate. The myosin head domain is followed by a neck domain consisting of six unevenly spaced consecutive IQ motifs representing light chain binding sites. The tail domain contains a C6H2-zinc binding motif and a region that specifically stimulates the GTPase-activity of Rho followed by a short stretch predicted to adopt a coiled-coil structure. Five alternatively spliced regions, one in the 5′-noncoding region, two in the myosin head and two in the tail domain, were noted. Analysis of myr 7 and myr 5 expression in different tissues revealed that myr 7 is expressed at high levels in developing and adult brain tissue whereas myr 5 is expressed only at moderate levels in embryonic brain tissue and at even further reduced levels in adult brain tissue. Myr 5 is, however, highly expressed in lung, liver, spleen and testis. Myr 7 is expressed in all brain regions and is localized in the cytoplasm of cell bodies, dendrites and axons. Myr 5 exhibits an overlapping, but not identical cellular distribution. Finally, a myr 7 fusion protein encompassing the GAP domain specifically activates the GTPase-activity of Rho in vitro, and overexpression of myr 7 in HtTA1-HeLa cells leads to inactivation of Rho in vivo. These results are compatible with a role for myr 7 (and myr 5) in regulating Rho activity in neurons and hence in regulating neuronal morphology and function.