In the present study digestive enzyme activities were studied in the rumen, intestine and faeces of dairy cows fed rations differing in starch source. Three total mixed rations were prepared for dairy cows with maize cob silage (MCS), sodium hydroxide-treated wheat (SHW) or rolled barley as starch source. The study was arranged as 3 × 3 Latin square design with 3 cows fistulated in the rumen, duodenum and ileum. The rations consisted of grassclover silage and maize silage (~60% of dry matter (DM)), rapeseed cake, soyabean meal, sugar beet pulp and 1 of 3 different starch sources MCS, SHW or rolled barley (~25% of DM). Samples from different parts of the digestive tract (rumen, duodenum, ileum) and faeces were collected and enzymatic activities of α-amylase, protease and lipase as well as their products content in fresh samples were estimated. When MCS replaced barley or SHW, it resulted in lower DM (2.61 vs 2.91 and 3.15%) and a higher ash content (30.99 vs 29.24 and 24.31%) in the ruminal fluid without affecting enzyme activities. Positive correlation between lipolytic and amylolitic activities in ruminal fluid was stated, which supported the hypothesis that amylolytic bacteria provide energy for lipolytic bacteria. So, the enzymes activities in the different parts of the digestive tract were not affected by the different starch sources.