The precise mechanisms governing the central distribution of macromolecules from the cerebrospinal fluid (CSF) to the brain and spinal cord remain poorly understood, despite their importance for physiological processes such as antibody trafficking for central immune surveillance as well as several ongoing intrathecal clinical trials. Here, we clarify how immunoglobulin G (IgG) and smaller single‐domain antibodies (sdAb) distribute throughout the whole brain in a size‐dependent manner after intrathecal infusion in rats using ex vivo fluorescence and in vivo 3D magnetic resonance imaging. Antibody distribution was characterized by diffusion at the brain surface and widespread distribution to deep brain regions along perivascular spaces of all vessel types, with sdAb accessing 4–7 times greater brain area than IgG. Perivascular transport involved blood vessels of all caliber and putative smooth muscle and astroglial basement membrane compartments. Perivascular access to smooth muscle basement membrane compartments also exhibited size‐dependence. Electron microscopy was used to show stomata on leptomeningeal coverings of blood vessels in the subarachnoid space as potential access points for substances in the CSF to enter the perivascular space. Osmolyte co‐infusion significantly enhanced perivascular access of the larger antibody from the CSF, with intrathecal 0.75 m mannitol increasing the number of perivascular profiles per slice area accessed by IgG by approximately 50%. Our results reveal potential distribution mechanisms for endogenous IgG, one of the most abundant proteins in the CSF, as well as provide new insights needed to understand and improve drug delivery of macromolecules to the central nervous system via the intrathecal route. This article is protected by copyright. All rights reserved