a) Introduction. The organisation and biophysical properties of the biological membrane surrounding fat globules in milk are poorly known and need further investigation to better understand their technological and biological functions. The milk fat globule membrane (MFGM) is the interface between the triacylglycerol core of milk fat globules and their aqueous environment. This membrane is important for the physical stability of fat globules and is involved in the mechanisms of milk fat globule digestion in the gastrointestinal tract of mammal newborns. The MFGM is essentially composed of polar lipids (of which 30% mol is milk sphingomyelin; MSM), cholesterol and membrane proteins. The lateral segregation of polar lipids characterised by high phase transition temperature (e.g. the MSM with Tm=34°C but also DPPC) has been revealed in the external bilayer of the MFGM and interpreted as the formation of “lipid rafts” at the surface of the milk fat globule (Lopez et al., 2010). The properties of rafts in the biological cells, and the interactions between cholesterol and sphingolipids such as sphingomyelin, have been the subject of many studies (Simons and Vaz, 2004). Increasing knowledge about the role of cholesterol in the physical characteristics of the high Tm lipid domains formed in the MFGM is of primary interest. Previous results indicated that the cholesterol strongly impacted on the lateral packing of the MSM in MSM/DOPC/cholesterol bilayers (Guyomarc’h et al., 2014). The objectives of this study were to investigate the role of cholesterol on the lipid domains formed in the MFGM, using Langmuir-Blodgett monolayers probed at the nanoscale using Atomic Force Microscopy (AFM) to characterise the structural details. b) Materials & methods. Investigations of lipid heterogeneities have been performed using a MFGM lipid extract (39% MSM, 32% PC, 24% PE, 3% PI, 3% PS). MFGM lipid extracts with 0 to 50 % mol. of cholesterol to the milk sphingomyelin (MSM) were prepared in methanol/chloroform then deposited at the air-water interface of a Langmuir trough containing PIPES buffer at 20°C. The lipids were compressed to 30 mN/m at 5 cm2/min, transferred at constant pressure onto freshly cleaved mica then dried in a dessicator for at least 3 days. Imaging was conducted with an MFP-3D Asylum Research AFM at room temperature. c) Results & discussion. Monolayers of MFGM lipids showed the formation of domains, which have been interpreted as the lateral segregation of high Tm lipids form the other lipids (polar lipids containing unsaturated fatty acids and forming the fluid phase surrounding the domains). The lipid domains formed in the absence of added cholesterol (Fig.1, left), had a µm-large size and protruded above the continuous phase by ~1.4 nm height step. As the proportion of cholesterol was increased (Fig. 1), the domains were clearly more numerous while decreasing both in area and in height step, the latter being only ~0.9 nm from 35% mol cholesterol onwards. This indicated that cholesterol scattered and fluidified the lipid domains into the continuous fluid phase (Milhiet et al., 2001). Langmuir compression isotherms were recorded and clearly evidenced the condensing effect of cholesterol onto the MFGM polar lipids, most probably the MSM (Simons and Vaz, 2004).