Transfection of cells with expression vectors is one of the most important tools used to assess the effects of receptor mutations on ligand-induced receptor sequestration. Most transfection methods give rise to transiently or stably transfected clones with a wide range of receptor expression levels that may also depend on the mutations made. It is, therefore, important to determine how the regulation of the receptors depends on their numbers per cell. In Chinese hamster ovary (CHO) and human embryonic kidney (HEK)-293 cells expressing high levels of B₂ kinin receptors, we observed poor sequestration indicated by <20% reduction in cell surface receptor number after 10 min of stimulation with 1 µM bradykinin (BK) compared with >70% in low-expressing cells. Whereas the rate of [³H]BK internalization (internalized [³H]BK in percentage of total bound [³H]BK) in low-expressing cells was independent of the ligand-concentration used, in high-expressing cells a strong rate decrease was observed with higher (>1 nM) concentrations. Lower ligand concentrations, however, led to internalization rates identical to those obtained in low-expressing cells. Transiently transfected HEK and COS-7 cells showed results similiar to those of stably high-expressing cells. Our results demonstrate the difficulty in determining the internalization pattern of (mutated) B₂ kinin receptors, and possibly of G protein-coupled receptors in general, using a sequestration assay in high-expressing cells or transiently transfected cells with high numbers of receptors per transfected cell. However, the receptor (mutant)-specific internalization rate can be measured, provided that the ligand concentrations used are below a threshold at which the internalization rate is still independent of the ligand concentration.