Data were collected from six experiments using duodenally cannulated Holstein dairy cows (88 combinations of cow and period) to evaluate the relationship between urinary purine metabolites and microbial N flow. Experiments evaluated the effects of dietary factors on microbial N production, which included 1) varying concentrations of ruminally degradable protein and nonstructural carbohydrates, 2) supplemental sources of protected amino acids, 3) grass silage treated with fibrolytic enzymes, 4) bacterial inoculation of corn silage, and 5) ruminal starch availability as affected by corn silages of varying maturity. The coefficient of determination for individual experiments that measured the relationship between microbial N flow and allantoin or uric acid excretion in urine ranged from 0.01 to 0.68 and 0.02 to 0.82, respectively. Across all experiments, the coefficients of determination between microbial N flow and allantoin or uric acid excretion in urine were r(2) = 0.002 and 0.11, respectively. Removal of data from one experiment improved the overall coefficient of determination between microbial N flow and urinary uric acid to r(2) = 0.32. Urinary allantoin excretion across experiments was negatively correlated with microbial N flow, but urinary allantoin excretion within experiments was positively correlated with microbial N flow. Uric acid excretion in urine was positively correlated with microbial N flow across and within experiments, except for one experiment. Our data demonstrate that uric acid excretion in urine can be used to predict microbial N production, except in early lactation, and that urinary allantoin excretion cannot be used to predict microbial N production accurately among cows at different stages of lactation.