Glucose-dependent insulinotropic polypeptide (GIP 1-42) is degraded by dipeptidyl peptidase IV (DPP IV), forming GIP 3-42. In mice, high concentrations of synthetic GIP 3-42 may function as a GIP receptor antagonist, but it is unclear whether this occurs at physiological concentrations. In COS-7 cells transiently transfected with the human GIP receptor, GIP 1-42 and 3-42 bind with affinities (IC₅₀) of 5.2 and 22 nM, respectively. GIP 1-42 was a potent agonist, stimulating cAMP accumulation (EC₅₀, 13.5 pM). GIP 3-42 alone had no effect. When incubated together with native GIP, GIP 3-42 behaved as a weak antagonist (IC₅₀, 92 and 731 nM for inhibition of cAMP accumulation elicited by 10 pM and 1 nM native GIP, respectively). In the isolated perfused rat pancreas, GIP 3-42 alone had no effect on insulin output, and only reduced the response to GIP (1 nM) when co-infused in >50-fold molar excess (IC₅₀, 138 nM). The ability of GIP 3-42 to affect the antihyperglycemic or insulinotropic actions of GIP 1-42 was examined in chloralose-anesthetized pigs given intravenous glucose. Endogenous DPP IV activity was inhibited to reduce degradation of the infused GIP 1-42, which was infused alone and together with GIP 3-42, at rates sufficient to mimic post-prandial concentrations of each peptide. Glucose, insulin and glucagon responses were identical irrespective of whether GIP 1-42 was infused alone or together with GIP 3-42. We conclude that although GIP 3-42 can weakly antagonize cAMP accumulation and insulin output in vitro, it does not behave as an antagonist in vivo.