The effect of altering supplementation frequency on host N balance and key N transactions in the ruminal ecosystem were monitored. Four ruminally fistulated beef steers (BW = 513 kg; SEM = 6.5) were used in a 2 x 2 crossover design with two periods and two supplementation frequency treatments. Supplementation frequencies were 2 and 7 d/wk. Steers were fed tallgrass prairie hay (73.1% NDF, 5.3% CP) ad libitum. Supplement (42% CP; DM basis) was fed at 0.36% BW/d to steers supplemented 7 d/wk. Steers supplemented 2 d/wk received the same amount of supplement per week, but it was equally split among the two supplementation events. Steers supplemented 7 d/wk had higher forage (P < 0.02) and total digestible OM intake (P < 0.06), total N intake, fecal N excretion, and N retention. Although both supplementation frequencies were characterized by positive N balance, the decrease in N retention in the steers supplemented 2 d/wk was due to higher (P < 0.01) urinary N loss. Ruminal fluid was sampled at 0, 2, 4, 6, 12, 24, 48, and 72 h after supplementation beginning on a day when both treatments were supplemented. Frequency x hour interactions (P < 0.02) were observed for ruminal N metabolism criteria. Counts of peptide- and AA-fermenting bacteria peaked at 2 h and returned to nadir by 12 h for steers supplemented 7 d/wk. Steers supplemented 2 d/wk peaked at 6 h with a greater population and returned to nadir at 48 h. Ruminal ammonia concentrations followed a similar trend. Specific activity of ammonia production was lower (P < or = 0.05) immediately after supplementation for steers supplemented 2 d/wk, but by 12 h was the same as for 7 d/wk steers. Ruminal peptides and free AA peaked at 2 h for steers supplemented 2 d/wk and were generally higher (P < or = 0.05) during the first 6 h compared with steers supplemented 7 d/wk. Total VFA concentration was not different (P = 0.35) due to supplementation frequency. Frequency x hour interactions (P < 0.01) were observed for all molar proportions of VFA. The molar proportion of acetate and acetate:propionate ratio were lower (P < 0.01) and the molar proportions of propionate and butyrate were higher for steers supplemented 2 d/wk from 4 h to 24 h. In conclusion, forage use and N balance improved with supplementation 7 d/wk, but supplementation 2 d/wk was associated with some desirable shifts in select ruminal events that may contribute to moderating potential negative impacts of supplementing infrequently.