Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK1R). The effects of sustained stimulation by high concentrations of SP on NK1R trafficking and Ca2+ signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca2+ signaling by wild-type NK1R (NK1Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK1R5K/R, C-terminal tail lysines; and NK1R10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca2+ ions. SP desensitized NK1Rwt, NK1R5K/R, and NK1R10K/R. However, NK1R5K/R and NK1R10K/R resensitized 4-8 fold faster than NK1Rwt by cycloheximide-independent mechanisms. NK1R325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK1Rwt, NK1RI?5K/R, and NK1RI?10K/R. After 4 h in SP-free medium, NK1RI?5K/R and NK1RI?10K/R recycled to the plasma membrane, whereas NK1Rwt remained internalized. SP induced ubiquitination of NK1Rwt and NK1RI?5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK1R10K/R was not ubiquitinated. Whereas SP induced degradation of NK1Rwt, NK1R5K/R and NK1R10K/R showed 50 diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK1R, which mediates its degradation and down-regulation.