The objectives of this experiment were to investigate the effects of two ruminally degradable protein (RDP) levels in diets containing similar ruminally undegradable protein (RUP) and metabolizable protein (MP) concentrations on ruminal fermentation, digestibility, and transfer of ruminal ammonia N into milk protein in dairy cows. Four ruminally and duodenally cannulated Holstein cows were allocated to two dietary treatments in a crossover design. The diets (adequate RDP [ARDP] and high RDP [HRDP]), had similar concentrations of RUP and MP, but differed in CP/RDP content. Ruminal ammonia was labeled with 15N and secretion of tracer in milk protein was determined for a period of 120 h. Ammonia concentration in the rumen tended to be greater (P = 0.06) with HRDP than with ARDP. Microbial N flow to the duodenum, ruminal digestibility of dietary nutrients, DMI, milk yield, fat content, and protein content and yield were not statistically different between diets. There was a tendency (P = 0.07) for increased urinary N excretion, and blood plasma and milk urea N concentrations were greater (P = 0.002 and P = 0.01, respectively) with HRDP compared with ARDP. Milk N efficiency was decreased (P = 0.01) by the HRDP diet. The cumulative secretion of ammonia 15N into milk protein, as a proportion of 15N dosed intraruminally, was greater (P = 0.003) with ARDP than with HRDP. The proportions of bacterial protein originating from ammonia N and milk protein originating from bacterial or ammonia N averaged 43, 61, and 26% and were not affected by diet. This experiment indicated that excess RDP in the diet of lactating dairy cows could not be efficiently utilized for microbial protein synthesis and was largely lost through urinary N excretion. At a similar MP supply, increased CP or RDP concentration of the diet would result in decreased efficiency of conversion of dietary N into milk protein and less efficient use of ruminal ammonia N for milk protein syntheses.