Atomic force microscopy (AFM) imaging was used to study swelling of individual bilayer graphene oxide (GO) flakes in water and ethanol vapors. We found that within 5 days after sample deposition the swelling of bilayered Hummers GO (HGO) in ethanol vapors disappears nearly completely, whereas the swelling in water remains not affected. Swelling can therefore be used as a sensitive indicator of rapid aging of bilayered HGO, which occurs both in air and under inert gas. The surprising ability of 5-days-aged bilayered HGO to swell in water but not in ethanol fits to the effects observed in μm-thick GO membranes after several years of aging. Remarkably, bilayered Brodie GO (BGO) maintains swelling in ethanol after storing it under the same conditions as HGO. Moreover, we demonstrate that AFM can be used to detect hole defects in individual GO sheets. The BGO bilayer swelling in ethanol vapors starts either on the flake edges or in very few points, forming ∼2 Å height fronts, which propagate slowly within interlayer spaces in all directions. The increase of the average distance between HGO sheets occurs simultaneously all over the flake, demonstrating a high abundance of hole defects. Our results imply that the permeation path length in HGO membranes must be significantly shorter compared to BGO, which is important to take into account in the modeling of membrane permeation.