G protein-coupled receptor kinases (GRKs) 2 and 3 (beta-adrenergic receptor kinases 1 and 2 (betaARK1 and -2)) mediate the agonist-dependent phosphorylation and uncoupling of many G protein-coupled receptors. These two members of the GRK family share a high degree of sequence homology and show overlapping patterns of substrate specificity in vitro. To define their physiological roles in vivo we have generated mice that carry targeted disruption of these genes. In contrast to GRK2-deficient mice, which die in utero (Jaber, M., Koch, W. J., Rockman, H., Smith, B., Bond, R. A., Sulik, K. K., Ross, J. JR., Lefkowitz, R. J. Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974-12979), GRK3 deletion allows for normal embryonic and postnatal development. GRK3 is expressed to a high degree in the olfactory epithelium, where GRK2 is absent. Here we report that cilia preparations derived from GRK3-deficient mice lack the fast agonist-induced desensitization normally seen after odorant stimulation. Moreover, total second messenger (cAMP) generation in these cilia preparations following odorant stimulation is markedly reduced when compared with preparations from wild-type littermates. This reduction in the ability to generate cAMP is evident even in the presence of nonodorant receptor stimuli (GTPgammaS and forskolin), suggesting a compensatory dampening of the G protein-adenylyl cyclase system in the GRK3 (-/-) mice in the olfactory epithelium. These findings demonstrate the requirement of GRK3 for odorant-induced desensitization of cAMP responses.