A high-performance liquid chromatography method with pre-column derivatization was tested and used in the analysis of partially separated 2,6-diaminopimelic acid (DAPA) in rumen bacteria, duodenal digesta and feeds incubated in vitro with rumen fluid. The samples of analyzed materials were hydrolyzed with 6M HC1 for 20 h at 104±2°C. DAPA was determined after pre-column derivatization with o-phthaldialdehyde (OPA) in the presence ethanethiol (ESH). Separation of converted DAPA was carried out using a reversed-phase column (Nova-Pak C-18, 4 µm, 250 x 4.6 mm I.D., Waters) by a binary gradient program and fluorescence or UV detection. The converted DAPA (as two peaks) was fluorescently monitored at an excitation wavelength of 229 nm, with 470 nm cutoff-filter (the retention times: 33.83±0.16 and 34.43±0.16 min), while the UV detector was set at 229 nm (retention times: 33.75±0.16 and 34.36±0.16 min). The DAPA peaks were completely resolved from interfering species in about 41 min. After 41 min, the column was re-equilibrated and cleaned, depending on the type of analyzed sample, for 9-14 min. The average analytical recoveries were 98.4±3.1% with fluorescence detection and 96.7±4.0% with UV detection for total DAPA. The low within and between run coefficients of variations, high recoveries and low detection (2.05 nmol/ml) and quantification (6.78 nmol/ml) limits, point to satisfactory precision, reproducibility, accuracy and sensitivity of the proposed method. The use of fluorescence detection and the sum of DAPA peaks give the method high precision and accuracy. The presented method enabled partial separation of DAPA stereoisomers, therefore this new HPLC procedure can also be used for investigations of DAPA metabolism within the rumen microbial ecosystem.