Long-term administration of histamine H₂ receptor (H₂R) antagonist (inverse agonist) induces up-regulation of H₂R in parietal cells, which may be relevant to rebound hypersecretion of gastric acid that occurs after withdrawal of treatment. The mechanisms underlying this effect are unknown. We hypothesized that the H₂R up-regulation could be related to receptor trafficking and used H₂R-green fluorescent protein (H₂R-GFP) to test the hypothesis. Human H₂R-GFP was generated and functionally expressed in HEK293 cells. Binding of the H₂R antagonist, [³H]tiotidine was performed to quantify the H₂R expression, and the H₂R-GFP was imaged in living cells by confocal and evanescent wave microscopy. The binding affinity of [³H]tiotidine was not significantly different between H₂R-GFP- and wild-type H₂R-expressing HEK293 cells, both of which had constitutive activity of adenylate cyclase. Visualization of H₂R-GFP revealed that the agonist induced H₂R internalization, and that the antagonist induced recycling of the internalized H₂R from the recycling endosome within 2 h. Long exposure to antagonist increased GFP fluorescence in the plasma membrane and also induced up-regulation of H₂R-GFP estimated by the binding assay, whereas long exposure to agonist enhanced degradative trafficking of H₂R-GFP. We examined whether the up-regulation reflected an increase in receptor synthesis. Treatment with antagonist did not augment H₂R mRNA, and subsequent inhibition of protein synthesis by cycloheximide had no effect on the H₂R up-regulation. These findings suggested that upon exposure to antagonist (inverse agonist), the equilibrium between receptor endocytosis and recycling is altered prior to H₂R up-regulation, probably via suppressing H₂R degradation.