This study was conducted to investigate the effect of dextrose, starch, NDF, and a carbohydrate (CHO) mix on utilization of ruminal ammonia in dairy cows. Four ruminally and duodenally cannulated Holstein cows (BW = 788 +/- 31 kg; 217 +/- 35 d in milk) were allocated to four treatments in a 4 x 4 Latin square design trial. Cows were fed an all alfalfa diet at 12-h intervals (DMI = 22.2 +/- 0.25 kg/d). Treatments were control, white oat fiber (NDF); corn dextrose (GLU); cornstarch (STA); and a CHO mix (25% of each): apple pectin, GLU, STA, and NDF (MIX). Carbohydrates were introduced intraruminally during feeding at 20% of dietary DMI. Ruminal ammonia was labeled with (15)N. Ruminal pH was the highest for NDF followed by STA and MIX and GLU (P < 0.001). Ruminal ammonia concentration and pool size were decreased by GLU and STA compared with NDF (P < 0.001 and P = 0.03, respectively). Acetate, isobutyrate, isovalerate, and total VFA concentration in the rumen were decreased (P = 0.009 to 0.001), and butyrate was increased (P < 0.001) by GLU compared with the other CHO. Microbial N flow to the duodenum was decreased (P < 0.05) by NDF compared with the other CHO, and the flow of microbial N formed from ammonia was greater for STA compared with GLU and NDF (P = 0.04 and 0.03, respectively). Urinary N loss was decreased (P = 0.05) by GLU and STA, but overall (feces plus urine) N losses were not affected (P = 0.73) by treatment. Milk urea concentration was lowered by GLU and STA compared with NDF and MIX (P = 0.002). The proportion of bacterial N synthesized from ammonia in the rumen was greater with STA than with NDF and MIX and was least for GLU (P = 0.02). Irreversible ammonia loss and flux were lower (P = 0.09 and 0.02, respectively) for GLU than for STA and NDF. As a percentage of the dose given, cumulative secretion of (15)N ammonia in milk protein was greater for STA than for GLU or NDF (P = 0.01 and 0.001, respectively). This experiment demonstrated that provision of readily fermentable energy can decrease ammonia concentrations in the rumen through decreased ammonia production (GLU), or through enhanced uptake of ammonia for microbial protein synthesis (STA). Rapidly fermentable energy in the rumen decreased ammonia production and flux, but the overall efficiency of ammonia utilization for milk protein synthesis was only increased by enhancing ruminal microbial ammonia uptake.