Eight rumen cannulated Simmental heifers (BW = 281.4 ± 18.5 kg) were randomly assigned to 1 of 4 experimental treatments to determine the effects of increasing levels of alfalfa hay on ruminal fermentation. Treatments tested were: a) total mixed ration with 10% barley straw as forage source (10BS), b) total mixed ration with 13% alfalfa hay as forage source (13AH), c) total mixed ration with 16% alfalfa hay as forage source (16AH) and d) total mixed ration with 19% alfalfa hay as forage source (19AH). Forages were coarsely chopped before their incorporation in total mixed ration. Diets were offered on an ad libitum basis, and formulated to be isocaloric (2.83 Mcal ME/kg DM) and isonitrogenous (14% CP on DM basis). After 2 weeks of diet adaptation, ruminal samples were taken immediately before feeding and at 4, 8, 12, 16, and 24 h after feeding on d1, d4 and d7 of the sampling week. Differences were analyzed by using the MIXED procedure of SAS. The model contained the fixed effects of treatment and the random effect of heifer. Day and hour were used as repeated measure. Intake of DM recorded during the sampling week was affected by treatment, being higher in heifers fed 16AH and 19AH than in heifers fed 10BS and 13AH (8.9 and 8 kg/d on average, respectively; P < 0.001). Heifers fed 19AH had a higher mean ruminal pH compared with the other treatments (6.74 vs. 6.55 on average; P = 0.02) and a higher total area under the pH curve (P = 0.01). Concentration of NH3-N (1.8 mg N/100mL) and total VFA (84 mM) were not affected by treatment (P > 0.10). Heifers fed 10BS presented the lowest proportion of acetate (P = 0.05) and the highest proportion of propionate (P = 0.04). Molar proportions of butyrate, valerate, isobutyrate and isovalerate were not affected by treatment (P > 0.10). Results indicate that the inclusion of alfalfa hay at 19% of total DM in a high-concentrate diet fed to growing heifers did not affect total VFA and NH3-N concentration, increased acetate and decreased propionate proportion, and increased mean ruminal pH, which can reduce the risk of ruminal acidosis.