Four rumen-cannulated dairy cows were fed with two levels of degradable protein (110 vs. 80% of requirements) and two type of carbohydrates (NDF/starch of 2.8 and 1.3) according to a 4x4 Latin square. Rumen contents were sampled at 0, 2.5 and 5h after feeding for DNA extraction. Bacterial and anaerobic fungal populations were studied by using TRFLP and ARISA respectively. Sampling time modified neither the microbial population structure nor the biodiversity indexes. Bacterial population composition was affected by the type of carbohydrate used but did not modify the bacterial diversity, suggesting a shift in the bacterial species profile but the maintenance of population structure. Bacterial clustering was also affected by the protein level, particularly in animals upplemented with starch. Anaerobic fungal population was also affected by the type of carbohydrate, and fibrous diet increased the number of amplicons (P<0.001). Dietary protein level did not modify the anaerobic fungal clustering, but high protein diets increased the number of amplicons (P<0.001). Animals fed the low protein diet supplemented with starch showed the lowest bacterial (34.6 vs. 37.2 fragments; P=0.01) and fungal (28.6 vs. 33.1 amplicons; P=0.05) diversity, and may be related to the highest efficiency of dietary N utilization observed in this experimental group. Keywords: arisa, fungi, rumen, trflp