Four ruminally fistulated Holstein heifers (BW = 385 +/- 6.2 kg) were used in a 4 x 4 Latin square experiment to determine the effect of feeding frequency on intake, water consumption, ruminal fermentation, and feeding and animal behavior. The treatments consisted of different feeding frequencies: a) once daily (T1); b) twice daily (T2); c) 3 times daily (T3); and d) 4 times daily (T4). Heifers were offered ad libitum access to concentrate and barley straw. Feeding frequency did not affect DMI (P >0.10), but water consumption tended to increase linearly as feeding frequency increased (P = 0.08). Average ruminal pH was not affected (P >0.10) by feeding frequency, but at 12 h after feeding ruminal pH was greater for T2 than for the other treatments. Total VFA concentration and VFA proportions were not affected (P >0.10) by feeding frequency, except valerate proportion, which increased linearly (P = 0.05) as feeding frequency increased. The concentration of ammonia-N was affected (P <0.05) cubically as feeding frequency increased (greatest for T3 = 9.3 mg of N/100 mL; lowest for T2 = 7.2 mg of N/100 mL). Feeding frequency had no effect on daily percentages of behavioral activities (P >0.05), except for observational behavior, for which there was a linear decrease as feeding frequency increased (P = 0.02). Heifers spent the same time on chewing activities, independent of feeding frequency. However, meal criteria tended to be affected (P = 0.07) by feeding frequency, with T2 (39.4 min) showing the longest intermeal interval. Total daily meal time, meal frequency, and meal size were not affected by feeding frequency (P >0.10), whereas meal length and eating rate showed cubic tendencies (P = 0.10 and P = 0.06, respectively) as feeding frequency increased. These results suggest that in the present experimental conditions, with heifers fed high-concentrate diets and with noncompetitive feeding, a smaller range of ruminal pH values was observed when feed was offered twice daily. Although heifers spent the same time on chewing activities, more stable ruminal conditions were probably achieved by feeding twice daily due to the rumination pattern, which was more constant during daytime in T2 than in T1. Moreover, when daytime and nighttime ruminating activity were analyzed separately, this activity was different in T1 (17.3 vs. 30.8%, respectively; P <0.05) but not in T2 (21.5 vs. 28.0%, respectively; P >0.05).