Intracellular trafficking of plant receptor kinases (PRKs) is a key step in regulation of cellular signaling. Our current knowledge in this field is based on systems that address signaling pathways affecting the whole cell. There are, however, signaling phenomena that add a further layer of complexity. In the Brassica self-incompatibility response, a single cell can adequately respond to two opposite stimuli: accepting cross-pollen and rejecting self-pollen simultaneously. To understand how PRK signaling can influence the coexistence of two seemingly exclusive states of the cell, we investigated the subcellular localization and internalization of the S-receptor kinase (SRK) involved in the self-incompatibility response of Brassica oleracea. Here, we describe the unusual subcellular distribution of SRK3, which localizes predominantly to intracellular compartments and to a much lesser extent to the plasma membrane. Using an anti-SRK antibody that fully substitutes for the natural ligand, we demonstrate that the interaction with the receptor takes place at the plasma membrane and is followed by SRK internalization in endosomes that are enriched in the SRK negative regulator Thioredoxin-h-like1.