Four multiparious Holstein cows fitted with ruminal and duodenal cannulae were in a 4 X 4 Latin square experiment to assess effects of increasing dietary protein on ruminal blood and duodenal traits, milk production, and nutrient digestion. Protein percents of 13, 15, 17, and 19 dry matter were achieved by rolled barley replaced with 0, 12, 24, and 36% rapeseed (canola) meal in pelleted concentrate mixtures. Increasing dietary protein concentration increased ruminal ammonia nitrogen, urea nitrogen in blood serum, and concentration of duodenal total, nonammonia, and feed nitrogen. Highest microbial nitrogen concentration was in cows fed the 15% protein diet; ruminal ammonia nitrogen was 6 mg/100 ml at this percent of protein. Cows fed 13% protein lost body weight (.43 kg/day), whereas those at 15, 17, and 19% protein gained .59, .38, and .59 kg/day. Efficiency of conversion of feed protein to milk protein declined, but digestibility of dry matter, organic matter, and nitrogen was improved with increasing dietary protein. From the traits measured, 15% crude protein appeared to be optimum for cows producing an average of 28 kg milk per day.