Bacterial predation by protozoa has the most deleterious effect on the efficiency of N use within the rumen, but differences in activity among protozoal groups are not completely understood. Two in vitro experiments were conducted to identify the protozoal groups more closely related with rumen N metabolism. Rumen protozoa were harvested from cattle and 7 protozoal fractions were generated immediately after sampling by filtration through different nylon meshes at 39°C, under a CO2 atmosphere to maintain their activity. Protozoa were incubated with 14C-labeled bacteria to determine their bacterial breakdown capacity, according to the amount of acid-soluble radioactivity released. Epidinium tended to codistribute with Isotricha and Entodinium with Dasytricha; therefore, their activity was calculated together. This study demonstrated that big Diplodiniinae had the greatest activity per cell (100 ng bacterial CP per protozoa and hour), followed by Epidinium plus Isotricha (36.4), small Diplodiniinae (34.2), and Entodinium plus Dasytricha (14.8), respectively. However, the activity per unit of protozoal volume seemed to vary, depending on the protozoal taxonomy. Small Diplodiniinae had the greatest activity per volume (325 ng bacterial CP per protozoal mm3 and hour), followed by big Diplodiniinae (154), Entodinium plus Dasytricha (104), and Entodinium plus Dasytricha (25.6). A second experiment was conducted using rumen fluid from holotrich-monofaunated sheep. This showed that holotrich protozoa had a limited bacterial breakdown capacity per cell (Isotricha 9.44 and Dasytricha 5.81 ng bacterial CP per protozoa and hour) and per protozoal volume (5.97 and 76.9 ng bacterial CP per protozoal mm3 and hour, respectively). Therefore, our findings indicated that a typical protozoal population (106 total protozoa/mL composed by Entodinium sp. 88%, Epidinium sp. 7%, and other species 4%) is able to break down ∼17% of available rumen bacteria every hour. Entodinium sp. is responsible for most of this bacterial breakdown (70 to 75%), followed by Epidinium sp. (16 to 24%), big Diplodiniinae (4 to 6%), and small Diplodiniinae (2 to 6%), whereas holotrich protozoa have a negligible activity (Dasytricha sp. 0.6 to 1.2% and Isotricha sp. 0.2 to 0.5%). This in vitro information must be carefully interpreted, but it can be used to indicate which protozoal groups should be suppressed to improve microbial protein synthesis in vivo.