AbstractIn Arabidopsis gravitropism is affected by two antagonistically interacting proteins, AGD12 (ADP‐RIBOSYLATION FACTOR GTPase‐ACTIVATING PROTEIN) and EHB1 (ENHANCED BENDING 1). While AGD12 enhances gravitropic bending, EHB1 functions as a negative element. To further characterize their cellular function, we analyzed the location of AGD12‐GFP and EHB1‐GFP fusion proteins in the root apex by confocal laser‐scanning microscopy after gravitropic stimulation. For this purpose, a novel method of microscopic visualization was developed with the objective and root axes aligned allowing an improved and comparable discernment of the fluorescence gradient across the columella. In vertical roots, both proteins were localized symmetrically and occurred preferentially in the outer layers of the columella. After reorienting roots horizontally, EHB1‐GFP accumulated in the upper cell layers of the columella, that is, opposite to the gravity vector. The gravity‐induced EHB1‐GFP asymmetry disappeared after reorienting the roots back into the vertical position. No such asymmetry occurred with AGD12‐GFP. Our findings reveal that after a gravitropic stimulus the cellular ratio between EHB1 and AGD12 is affected differently in the upper and lower part of the root. Its impact as a significant signaling event that ultimately affects the redirection of the lateral auxin flux toward the lower site of the root is discussed.