The CLA content and isomer profile were characterized among two subclasses of the native milk fat globules. The latter corresponded to two fractions obtained by a microfiltration process using Spring mixed milk, leading to small fat globules (SFG; Click to view the MathML source) in the microfiltrates and large ones (LFG; Click to view the MathML source) in the retentates. Relatively, SFG always contained more CLA than the LFG originating from the same mixed milk, though discrepancies among different milk samples were observed. The main CLA isomer was the cis-9, trans-11, the content of which tended to increase when the native milk fat globule size decreased (from 82.2% to 87.3% of total CLA isomers). Consequently, the SFG contained less trans-12, trans-14; trans-11, trans-13; trans-11, cis-13 and trans-8, cis-10 isomers than the corresponding LFG. However, the relative variation of some isomers between small and large fat globule from the same milk varied depending on milk origin and the potential of fat globule fractionation for influencing the content and distribution of CLA seems to be limited.