This study investigates 5-hydroxytryptamine 4 (5-HT₄) receptor binding in the minipig brain with positron emission tomography (PET), tissue homogenate-binding assays, and autoradiography in vitro. The cerebral uptake and binding of the novel 5-HT₄ receptor radioligand [¹¹C]SB207145 in vivo was modelled and the outcome compared with postmortem receptor binding. Different models for quantification of [¹¹C]SB207145 binding were evaluated: One-tissue and two-tissue compartment kinetic modelling, Logan arterial input, and three different reference tissue models. We report that the pig autoradiographic 5-HT₄ receptor distribution resembles the human 5-HT₄ receptor distribution with the highest binding in the striatum and no detectable binding in the cerebellum. We found that in the minipig brain [¹¹C]SB207145 follows one-tissue compartment kinetics, and the simplified reference tissue model provides stable and precise estimates of the binding potential in all regions. The binding potentials calculated for striatum, midbrain, and cortex from the PET data were highly correlated with 5-HT₄ receptor concentrations determined in brain homogenates from the same regions, except for hippocampus where PET-measurements significantly underestimate the 5-HT₄ receptor binding, probably because of partial volume effects. This study validates the use of [¹¹C]SB207145 as a promising PET radioligand for in vivo brain imaging of the 5-HT₄ receptor in humans.