Abstract: The aim of this study was to evaluate the effect of daily supplementation of a devitalized strain of Saccharomyces cerevisiae on milk yield and composition of Friesian-Holstein lactating cows. Average Days In Milk (DIM) and Body Weight (BW) of cows were 98±24 days and 619±13.2 kg. Prior to the experiment, the effects of the yeast culture added to the diet were investigated in 2 in vitro experiments. In the first experiment, 6 samples of rumen fluid were used to determine the DM, CP and NDF degradability (IVDMD, IVCPD, IVNDFD) of Total Mixed Ration (TMR) at 0 and 48 h with the addition of 0 g (Y0) and 1 g (Y1) of yeast culture. In the second experiment, free, total and microbial ATP production were used for the evaluation of microbial activity in rumen fluids after 0, 8, 24 and 48 h incubation. In the 4 weeks trial, 10 multiparous cows were assigned to a Control diet (C) and 10 to the experimental diet (YC = C+35 g/cow/day of yeast culture). TMR on a Dry Matter (DM) basis consisted of corn silage (42.3%), ryegrass hay (14.1%), corn meal (22.1%), soybean meal (17.9%) and a mineral/vitamins mix (3.6%). Crude Protein (CP) and ENl were 14.6% and 1.56 Mcal kg-1 DM, respectively. Cows were individually fed once daily and milked at 7 and 19 h. In vitro experiments showed that the addition of 1 g YC to the rumen fluid increased IVCPD and IVNDFD after 48 h of incubation but did not influence IVDMD. Free, total and microbial ATP values showed a continuous decrease during the 48 h of the trial but the addition of YC proved to be effective in slowing the decrease. Milk yield (31.9 and 33.4 kg day-1, for the C and YC group), 4% fat corrected milk (FCM: 30.3 and 31.7 kg day-1) and energy corrected milk (ECM: 32.9 and 34.5 kg day-1) were significantly different between groups. Dry Matter Intake (DMI), milk fat and protein percentage were similar in both groups. Feed efficiency (ECM kg-1 DMI) was higher in YC group than in C.