Six lactating Holstein cows fitted with rumen and T-type duodenal cannulas were used in a crossover design to examine effects of yeast culture supplement on production parameters, rumen fermentation, and flow of N to the duodenum. Treatments were control and control plus 10 g/d of yeast culture. Dry matter intake was greater, and milk production tended to be higher, for cows supplemented with yeast culture, but milk composition was not affected. Rumen pH was not affected by yeast culture, but peak lactic acid concentration decreased from 1.93 to 1.73 mM. Rumen fluid acetate:propionate ratio, dilution rate (percentage per hour), and ammonia N concentration (milligrams per deciliter) were 2.28, .12, and 10.7 and 2.04, .13, and 9.6 for control cows and for cows supplemented with yeast culture, respectively. Although numbers of fiber-digesting bacteria were not affected by yeast culture, DM disappearance of wheat straw tended to be higher at 12 and 24 h, and CP and ADF digestibilities were greater. Duodenal NAN flow tended to be higher in cows supplemented with yeast culture because of higher bacterial N flow. Duodenal AA profile and flow of Met were significantly affected by yeast culture supplementation. The results suggest that yeast culture may alter the AA profile of bacterial protein.