We examined the regulatory mechanisms of the affinity of Gq protein-coupled histamine H₁-receptors for histamine after histamine pretreatment in intact human U373 MG astrocytoma cells. In control cells, the displacement curves for histamine against the binding of 5 nM [³H]mepyramine, a radioligand for H₁-receptors, showed the presence of two binding sites for histamine, that is, high and low affinity sites. Pretreatment with 0.1 mM histamine for 30 min at 37°C induced a significant reduction in the percentage of high affinity sites for histamine and a concomitant increase in the percentage of low affinity sites with no change in their pIC₅₀ values. These histamine-induced changes were insensitive to 30 μM KN-62, an inhibitor of Ca²⁺/calmodulin-dependent protein kinase II, but they were completely inhibited either by 0.4 mM ZnCl₂, an inhibitor of G protein-coupled receptor kinases (GRKs), or under hypertonic conditions, where clathrin-mediated endocytosis is known to be inhibited. These results suggest that histamine-induced conversion of high to low affinity sites for histamine is predominantly regulated by GRK/clathrin-mediated internalization of H₁-receptors in human astrocytoma cells.