Two rumen-fistulated Holstein heifers were fed three protein-free semipurified diets: 70% roughage, 30% concentrate; 50% roughage, 50% concentrate; and 30% roughage, 70% concentrate. Wheat straw was the roughage, and the concentrate was a pelleted mixture of corn starch, dextrose, fat, urea, vitamins, and minerals. Each animal received the three dietary treatments in a randomized complete block design. On days 1, 3, and 5 following a 21-day adaptation, rumen fluid samples were taken before feeding and at 1, 2, 3, 4, and 5 h after feeding. Total protozoa concentrations for low, medium, and high concentrate diets were 1.5, 2.5, 4.1 X 10(5)/ml. Dasytricha concentrations were not affected by diet, but Entodinium, Diplodinium, Isotricha, Epidinium, and Ophryoscolex increased with increases of the concentrate proportion of the diets. Entodinium, the predominant protozoa with all diets, increased as concentrate proportion of the diet increased. In a second experiment the two heifers from Experiment 1 were fed semipurified diets that contained either urea or soybean meal as the nitrogen source. There were larger populations of Dasytricha and Isotricha when the diet contained soybean meal, and larger populations of Charon, Entodinium, and total protozoa when the diet contained urea. In both experiments, holotrichs were highest 1 to 2 h after feeding, and entodiniomorphs were highest before feeding and 4 to 6 h after feeding. An active protozoan population can exist in the rumen of cattle fed diets virtually devoid of natural protein, and protozoa are influenced by energy and nitrogen source.