The aim of this study was to examine the expression of G protein-coupled receptor (GPR)35 in human invariant natural killer T (iNKT) cells and to determine the functional effects induced by selective activation of this receptor. RT-PCR analysis showed that both human iNKT cells and resting PBMC expressed GPR35: GPR35 protein resulted mostly localized in the plasma membrane, while it internalized in punctate intracellular structures following specific receptor activation (Western blot and immunofluorescence/confocal microscopy analysis). The specific activation of GPR35 by selective receptor agonists kynurenic acid (KYNA)] or 1,4-dihydro-5-(2-propoxyphenyl)-7H-1,2,3-triazolo 14,5-d]pyrimidine-7-one (zaprinast)] functionally correlated with a significant reduction in IL-4 release from alpha-galactosylceramide (alpha-GalCer)-activated human iNKT cells, and this effect resulted mediated by pertussis toxin (PTX)-sensitive Gi/o proteins. In conclusion, our results demonstrate that human iNKT cells express GPR35 functionally active in reducing IL-4 release. (C) 2010 Elsevier Inc. All rights reserved.