-Caseins, major proteins in bovine milk, are structured in micelles within which the detailed casein organisation remains unclear. We have used limited proteolysis to find the most exposed regions of the caseins within the micelle. Under hydrolysis conditions, only 9% of colloidal calcium and 15.6% of the colloidal inorganic phosphate were removed from the micelle after 4 h. The released peptides during tryptic hydrolysis were separated from the residual micelle by ultracentrifugation. They were subsequently analysed and characterised by reversed-phase HPLC coupled online with electrospray ion source mass spectrometry (ESI-MS). After 4 h of hydrolysis, the p-casein had completely disappeared while about 40% of us,-casein and 37% of x-casein remained undigested. The initial distribution, i.e., 91 % caseins and 9% peptides prior to hydrolysis, was modified after hydrolysis to give 64% peptides remaining within the micelles together with 13% caseins and 22% of the peptides produced were released from the micelles. Among the 61 peptides identified, 34 arose from p-casein, 16 from us1-casein, II from u s2 -casein and none from x-casein. For the latter casein, the failure ta detect peptides was due to its lower concentration in the large micelles cornpared with that of the P and us1-caseins. The peptides derived from p-casein were mainly released from the micelle, while peptides from bath u s1 -and u s2 -caseins were preferentially retained within the residual micelles, suggesting that ail the caseins are accessible ta trypsin but are differentially involved in the micellar framework. Our results suggest that us-caseins play a significant role in the micellar cohesion via salt binding with colloidal calcium phosphate and hydrophobie interaction, with itself and with the other caseins. © Inra/Elsevier, Paris.casein / micelle / Iimited proteolysis / trypsin / reversed-phase chromatography / mass spectrometry / peptide / accessibility Résumé -Sites privilégiés de coupure sur les caséines bovines majeures par trypsinolyse des micelles. Les caséines, constituants majeurs des protéines du lait de vache, sont présentes sous forme de micelles dont l'organisation interne est encore mal connue. Nous avons utilisé la protéolyse limitée pour déterminer les régions des caséines les plus accessibles au sein de la micelle. Dans les conditions utilisées, seuls 9 % du calcium colloïdal et 15,6 % du phosphate inorganique colloïdal sont * Correspondence and reprints: E-mail: jleonil@labtechno.roazhon.inra.fr 472 V. Gagnaire, J. Léonil éliminés de la micelle après 4 h d'hydrolyse. Afin de différencier les peptides qui sont libérés de la micelle de caséine au cours de l'hydrolyse par la trypsine de ceux qui restent situés à l'intérieur, nous avons ultracentrifugé les hydrolysats avant de caractériser les peptides. Leur caractérisation était ensuite réalisée par couplage direct entre la CLHP de phase inverse et la spectrométrie de masse à source d'ionisation electrospray. Après 4 h d'hydrolyse, la caséine~avait complètement dispar...