This investigation was conducted to quantify the purine excretion of cows and to evaluate milk allantoin as an estimator of synthesis of microbial protein in the rumen. Concentrations in plasma of allantoin and uric acid and their excretion in urine and milk were determined using 16 Holstein cows at 21 to 315 d of lactation and yielding 8 to 36 kg/d of milk. The mean daily intake of a 55:45 roughage and concentrate diet was 16.3 kg of DM with 106 MJ of NEL and 13.8 to 16.0% CP. Total urine was collected over 24 h, plasma samples collected four times, and milk samples collected twice on the same day. Analyses of purine metabolites were performed with HPLC. On average, excretion of allantoin in urine and milk was about 294 and 4.1 mmol/d for allantoin and 35 and 1.1 mmol/d for uric acid. Excretion of allantoin in milk was correlated to concentration in plasma (r = .84). As milk yield increased, percentage of milk allantoin increased from .6 to 2.4% of total allantoin excretion (r = .85). The correlations of NEL intake with allantoin excretion in milk (r = .80) and in urine (r = .84) were similar. Daily milk yield was correlated with plasma allantoin (r = .78) and milk allantoin excretion (r = .95). We concluded that determination of milk allantoin is a useful, noninvasive method to monitor rumen microbial protein synthesis.