Means analyses were used to compare estimates of crude protein (CP) degradation (%), true organic matter (OM) digestion (%), efficiency of microbial synthesis (g N kg-1 OM truly digested) and microbial nitrogen (N) flow (g d-1) using diaminopimelic acid (DAPA) and purines as microbial markers for 12 in vitro and four in vivo experiments. In vitro experiments included 268 determinations and in vivo experiments included 80 determinations of microbial activity by each marker. In vitro estimates of ruminal CP degradation, true OM digestion, efficiency of microbial synthesis and microbial N flow by purines (60.5, 52.4, 33.1 and 1.3) were lower (P < 0.05) than estimates by DAPA (69.9, 55.0, 36.1 and 1.4). Coefficients of variation of estimates of ruminal CP degradation, true OM digestion, efficiency of microbial synthesis and microbial N flow determined by purines (22.0, 12.4, 17.0 and 22.4) or DAPA (22.2, 12.3, 14.2 and 20.5) were similar for in vitro experiments. In vivo estimates of ruminal CP degradation, true OM digestion and microbial N flow by purines (57.2, 53.1 and 137.2) were lower (P < 0.05) than estimates by DAPA (65.9, 61.1 and 162.2). Estimates of efficiency of microbial synthesis determined using purines (28.4) and DAPA (27.9) were similar. Coefficients of variation for estimates of ruminal CP degradation, true OM digestion, efficiency of microbia l synthesis and microbial N flow determined by purines (21.4, 23.8, 38.6 and 32.4) and DAPA (16.5, 16.7, 37.3 and 33.9) were similar for in vivo experiments. Estimates of CP degradation exceeded 90% on 26 occasions when DAPA was used as the marker, compared with seven occasions when purines were used. Using purines as the microbial marker resulted in lower microbial activity and similar coefficients of variation compared with DAPA. Unrealistic values of CP degradation were observed more frequently using DAPA as the microbial marker.