Free fatty acid receptor 1 phosphorylation sites were studied using mutants, including a) a mutant with T215V in the third intracellular loop (3IL), b) another with changes in the carboxyl terminus (C-term): T287V, T293V, S298A, and c) a mutant with all of these changes (3IL/C-term). Agonist-induced increases in intracellular calcium were similar between cells expressing wild-type or mutant receptors. In contrast, agonist-induced FFA1 receptor phosphorylation was reduced in mutants compared to wild type. Phorbol ester-induced FFA1 receptor phosphorylation was rapid and robust in cells expressing the wild-type receptor and essentially abolished in the mutants. Agonist-induced ERK 1/2 phosphorylation and receptor internalization were decreased in cells expressing the mutant receptors compared to those expressing the wild-type receptor. Our data suggest that the identified sites might participate in receptor phosphorylation, signaling, and internalization.