p21-activated kinases (PAK) are involved in the control of cytoskeleton dynamics and cell cycle progression. Here we report the characterization of a new mammalian PAK3 mRNA that contains a 45-bp alternatively spliced exon. This exon encodes for 15 amino acids that are inserted in the regulatory domain, inside the autoinhibitory domain but outside the Cdc42 and Rac interactive binding domain. The transcript of the 68-kDa new isoform named PAK3b is expressed in various areas of the adult mouse brain. In contrast to PAK3 without the exon b (PAK3a), whose basal kinase activity is weak in resting cells, PAK3b displays a high kinase activity in starved cells that is not further stimulated by active GTPases. Indeed, we demonstrate that the autoinhibitory domain of PAK3b no longer inhibits the kinase activity of PAK3. Moreover, we show that the 15-amino acid insertion within the autoinhibitory domain impedes the ability of PAK3b to bind to the GTPases Rac and Cdc42 and changes its specificity toward the GTPases. Altogether, our results show that the new PAK3b isoform has unique properties and would signal differently from PAK3a in neurons.