Human milk fat globules (HMFGs) are considered to constitute a triglyceride-rich source of fat and energy. However, milk contains lipid particles at different sizes ranging from tens of micrometers to less than 1 microm. In particular, the physical, chemical, and biological properties of submicron sized particles are poorly described. Individual HMFGs were analyzed using laser trapping confocal Raman spectroscopy, and their chemical signature was obtained and compared to 1, 5, and 10 microm globules. Significant differences in both lipid composition and relative lipid content were found between the classes of particles with different diameters. A strong Raman peak at 1742 cm(-1) corresponding to the triacylglycerol core was detected in the 5 and 10 microm diameter globules, whereas in the smaller HMFGs no detectable peak was found. In addition, the submicron particles produced Raman signals consistent with large quantities of unsaturated fatty acids. Moreover, cis and trans isomers of unsaturated fatty acids were found to be unequally distributed between large and small milk fat globules. Interestingly, trans unsaturated fatty acids were found only in 1 and 5 microm globules although more prominent in the 5 microm diameter range. This is the first evidence for size related differential lipid composition of various diameter classes of HMFGs. The results suggest that the milk fat globule size distribution determines milk lipid composition. In addition, large portions of the HMFGs are secreted into milk conspicuously not for fat delivery. Thus, small HMFGs may offer novel metabolic and nutritional functions.