G protein-coupled receptor kinases (GRKs) are a family of intracellular proteins that desensitize and regulate the responsiveness of G protein-coupled receptors (GPCRs). In the present study, we assessed the contribution of GRK6 to the regulation and responsiveness of the G protein-coupled mu-opioid receptor (microOR) in response to morphine in vitro and in vivo using mice lacking GRK6. In cell culture, overexpression of GRK6 facilitates morphine-induced beta-arrestin2 (betaarrestin2) recruitment and receptor internalization, suggesting that this kinase may play a role in regulating the microOR. In vivo, we find that acute morphine treatment induces greater locomotor activation but less constipation in GRK6 knockout (GRK6-KO) mice compared to their wild-type (WT) littermates. The GRK6-KO mice also appear to be "presensitized" to the locomotor stimulating effects induced by chronic morphine treatment, yet these animals do not display more conditioned place preference than WT mice do. Furthermore, several other morphine-mediated responses which were evaluated, including thermal antinociception, analgesic tolerance, and physical dependence, were not affected by ablation of the GRK6 gene. Collectively, these results suggest that GRK6 may play a role in regulating some, but not all morphine-mediated responses. In addition, these findings underscore that the contribution of a particular regulatory factor to receptor function can differ based upon the specific cell composition and physiology assessed, and illustrate the need for using caution when interpreting the importance of interactions observed in cell culture.