A study was undertaken to investigate the effects of farming systems (organic vs. conventional), diet (hay/concentrate vs. pasture) and their interaction on milk yield, gross composition and fatty acid (FA) profile of dairy cows bred in mountainous areas. For this purpose four dairy farms (two organic and two conventional) were chosen in the alpine territory of Aosta Valley (NW Italy); individual milk yield was recorded daily and bulk milk samples were collected monthly from February to September 2007 to cover dietary variations. Higher levels of milk production (P<0.05) and lower milk protein amounts (P<0.01) were observed in the organic farms with respect to the conventional ones, while no significant differences were noticed in milk fat and lactose contents and in somatic cell count. Concerning fatty acids, only small differences were detected between organic and conventional milk and such differences seemed to be related mainly to the stabled period. Diet affected almost all variables studied: pasture feeding provided a significant improvement in the fatty acid composition in both organic and conventional systems leading to lower hypercholesterolemic saturated fatty acids, higher mono- and polyunsaturated fatty acids and conjugated linoleic acid amounts (P<0.001).