The subcellular distribution of lipids in human hair was investigated to better understand their role in water permeability. Unlike stratum corneum where lipids are organized under a precisely ordered continuous structure, the removal of free lipids in hair does not lead to an increase of water permeability. Esterified and CH2-enriched molecules were tracked at a 10 nm resolution by infrared nanospectroscopy (atomic force microscopy coupled to infrared spectroscopy, AFMIR). Free and bound lipids in the 25 nm thick intercellular spaces were directly detected for the first time, further substantiating the potential of AFMIR to study complex biomaterials. We observed that they were mostly found accumulated in some regions of the external cuticle layers, as "hotspots" in nonkeratinous portions of the internal cortex, and that they do not seem to modulate much the water exchanges due to their discrete distribution throughout the hair fiber.