Alterations in brain cholesterol homeostasis have been broadly implicated in neurological disorders. Notwithstanding the complexity by which cholesterol biology is governed in the mammalian brain, excess neuronal cholesterol is primarily eliminated by metabolic clearance via cytochrome P450 46A1 (CYP46A1). No methods are currently available for visualizing cholesterol metabolism in the living human brain; therefore, a noninvasive technology that quantitatively measures the extent of brain cholesterol metabolism via CYP46A1 could broadly affect disease diagnosis and treatment options using targeted therapies. Here, we describe the development and testing of a CYP46A1-targeted positron emission tomography (PET) tracer, 18F-CHL-2205 (18F-Cholestify). Our data show that PET imaging readouts correlate with CYP46A1 protein expression and with the extent to which cholesterol is metabolized in the brain, as assessed by cross-species postmortem analyses of specimens from rodents, nonhuman primates, and humans. Proof of concept of in vivo efficacy is provided in the well-established 3xTg-AD murine model of Alzheimer's disease (AD), where we show that the probe is sensitive to differences in brain cholesterol metabolism between 3xTg-AD mice and control animals. Furthermore, our clinical observations point toward a considerably higher baseline brain cholesterol clearance via CYP46A1 in women, as compared to age-matched men. These findings illustrate the vast potential of assessing brain cholesterol metabolism using PET and establish PET as a sensitive tool for noninvasive assessment of brain cholesterol homeostasis in the clinic.