The rates of production of protozoa in the rumen of buffalo calves have been estimated using a single-injection isotope dilution technique. The calves were fed at 2 levels of crude protein, namely, 13% lower and 19% higher than that recommended by the National Research Council. The animals were given their rations at 2-h intervals for 3 weeks. Thereafter 14C-labelled rumen protozoa were injected into the rumen of each calf in a single injection. Samples of the rumen liquor were drawn at various time-intervals for 10 h and were analysed for the concentration of protozoa and radioactivity. The decline in the specific radioactivity of protozoa cells in the rumen as a function of time was used for calculating half life (t½). A mathematical equation was used to calculate the rate of production of protozoa. The average t½ was 1077 min for both groups and the rates of production of protozoa were 73·9 and 92·1mg/min in groups fed on low and high planes of crude protein respectively, showing that the production of protozoa is significantly (P < 0·01) higher in animals consuming a ration high in crude protein.