Microbial rDNA sequences have been proposed as potential internal markers to determine microbial synthesis in the rumen. The objective of this experiment was to compare values of microbial growth determined using ¹⁵N as external marker with concentrations of microbial DNA in fermenters, and to assess if both procedures detected similar differences between diets and solid (SOL) and liquid (LIQ) digesta phases. Four Rusitec vessels were used in a crossover trial with 2 14-d periods. In each period, 2 fermenters received a 50:50 alfalfa hay:concentrate diet (MC) and 2 were fed a 15:85 barley straw:concentrate diet (HC). A solution of ¹⁵NH₄Cl was infused for 5 d before taken samples of SOL and LIQ digesta and isolation of bacterial pellets from both digesta phases to estimate microbial protein synthesis. Samples of SOL and LIQ digesta were simultaneously taken for DNA extraction and analysis of concentrations of total bacterial and protozoal DNA by quantitative PCR. Total microbial N (TMN) was calculated from the ¹⁵N enrichment in digesta and isolated bacterial pellets, and total microbial DNA (TMDNA) was calculated as the sum of bacterial DNA and protozoal DNA in both digesta fractions. There were no diet × digesta phase interactions (P > 0.05) with any marker. Both TMN and TMDNA were greater (P < 0.001) in MC-fermenters than in HC-fermenters (1.5 and 2.0 times greater for TMN and TMDNA, respectively). Values of TMN were greater (P = 0.004) in SOL than in LIQ digesta (108 and 89.7 mg N, respectively), whereas the opposite was found for TMDNA (3.37 and 13.1 mg DNA, respectively). There was no difference between diets (P > 0.05) in the contribution of SOL digesta to TMN (53.8 and 55.7% for MC and HC diets, respectively), but contribution of SOL digesta to TMDNA was greater in MC than in HC diet (P = 0.039; 24.5 and 11.5%, respectively). There was no relationship (P > 0.05) between TMN and TMDNA values, but a significant relationship was observed when only values in the LIQ digesta were considered (P = 0.024; r = 0.821). In summary, both markers detected similar differences between diets, but not between digesta phases.