Our published studies show that the distribution of the AT₁ receptor (AT₁R), expressed as a yellow fluorescent fusion protein (AT₁R/EYFP), is altered upon cellular treatment with angiotensin II (Ang II) or coexpression with intracellular Ang II. AT₁R accumulates in nuclei of cells only in the presence of Ang II. Several transmembrane receptors are known to accumulate in nuclei, some as holoreceptors, others as cleaved receptor products. The present study was designed to determine whether the AT₁R is cleaved prior to nuclear transport. A plasmid encoding a rat AT₁R labeled at the amino-terminus with ECFP and at the carboxy-terminus with EYFP was employed. Image analyses of this protein in COS-7 cells, CCF-STTG1 glial cells and A10 vascular smooth muscle cells show the two fluorescent moieties to be largely spatially colocalized in untreated cells. Ang II treatment, however, leads to a separation of the fluorescent moieties with yellow fluorescence accumulating in more than 30% of cellular nuclei. Immunoblot analyses of extracts and conditioned media from transfected cells indicate that the CFP domain fused to the extracellular amino-terminal AT₁R domain is cleaved from the membrane and that the YFP domain, together with the intracellular cytoplasmic carboxy-terminus of the AT₁R, is also cleaved from the membrane-bound receptor. The carboxy-terminus of the AT₁R is essential for cleavage; cleavage does not occur in protein deleted with respect to this region. Overexpressed native AT₁R (non-fusion) is also cleaved; the intracellular 6 kDa cytoplasmic domain product accumulates to a significantly higher level with Ang II treatment.