Recently, we have shown that the delta subunit of the cGMP phosphodiesterase (PDE delta) interacts with the retinitis pigmentosa guanine regulator (RPGR). Here, using the two-hybrid system, we identify a member of the Arf-like protein family of Ras-related GTP-binding proteins, Arl3, that interacts with PDE delta. The interaction was verified by fluorescence spectroscopy and co-immunoprecipitation. Arl3 features an unusually low affinity for guanine nucleotides, with a KD of 24 nM for GDP and 48 microM for GTP. Fluorescence spectroscopy shows that PDE delta binds and specifically stabilizes the GTP-bound form of Arl3 by strongly decreasing the dissociation rate of GTP. Thus, PDE delta is an effector of Arl3 and could provide a novel nucleotide exchange mechanism by which PDE delta stabilizes Arl3 in its active GTP-bound form.