Diurnal variation in the yield of microbial protein from the rumen may limit the use of urinary purine derivatives (PD) to estimate microbial protein yield from the rumen when assessed using spot samples. Chen, Grubic, Ørskov and Osuji (1992) found only small differences in the urinary PD excretion in young steers, and even less variation when PD excretion was estimated as a ratio to urinary creatinine. Lactating dairy cows have a much higher feed intake than steers and as a consequence may exhibit greater diurnal variation in urinary PD excretion. An examination of data from three experiments was undertaken to assess the effects of diurnal variation in rumen microbial protein yield on urinary PD excretion.