Three experiments in a 4 x 4 Latin square design were carried out on four rams fitted with rumen cannulas to investigate the effect of varying sources and levels of fat addition on microbial protein net synthesis (MN) in sheep fed a concentrate-based diet (60% of concentrate and 40% of meadow hay). In the 1^st experiment addition of rape seed oil (RSO) was tested, linseed oil (LSO) was added in the 2^nd, and tallow (TAL) in the in 3^rd experiment. Treatments in all experiments were similar: a control group without fat addition and experimental groups with 4, 8 and 10% of fat in DM of the diet. Microbial production in the rumen, purine derivatives in urine, ruminal ammonia, pH, fatty acids in the rumen were measured. In all cases fat addition resulted in decreased MN, however, 8% fat supplementation had the least deleterious effect on M N production. Fat addition significantly affected purine derivatives excretion. LSO as the supplemental fat led to a decrease in N ammonia concentration from 7.8 mmol/L in the control group to 6.39 mmol/L in the group that received 8% LSO, whereas a significant increase to 9.00 mmol/L was observed when 10% LSO in DM was fed. Ruminal fluid pH was not altered by treatments. In all of the experiments, the increased fat content in the diets decreased the molar percentage of acetic acid. Supplementation of the diet with LSO and TAL resulted in an increase of the propionic acid level (P<0.05).