Virginiamycin is an antibiotic active against grampositive bacteria in the alimentary tract, which is also suitable for supplementation of diets of growing and finishing ruminants. The aim of this work was to specify the effect of virginiamycin on some parameters of rumen fermentation in vitro with inoculi taken from wethers adapted or non-adapted to the virginiamycin intake. Incubations were performed anaerobically at 39 degrees C in serum bottles closed with Bunsen valves. Virginiamycin was added at 0 or 10 mg/l to the rumen fluid diluted with McDougall buffer. Virginiamycin significantly decreased production and utilization of lactic acid, production of methane and decomposition of casein when rumen fluid was taken from non-adapted wethers. Most of its effects disappeared when rumen fluid was sampled from wethers adapted to the virginiamycin intake (100 mg per head daily for 2 months). Adaptation of wethers to virginiamycin was further confirmed by analyses of the rumen fluid which was used for inoculation of in vitro cultures. Molar percentages of acetate, propionate, butyrate and valerate were the same before and after the adaptation. Therefore it can be concluded that the effects of virginiamycin on rumen parameters are not stable and its addition to ruminant diets cannot be recommended, with exception of the milk nutrition period. In the last experiment the stability of virginiamycin in the rumen fluid of adapted wethers was investigated. The rumen fluid was diluted with buffer containing glucose and virginiamycin (10 mg/l, final cone.) and incubated in the LF2 fermenter at pH = 6.5. Samples taken at 0, 3, 6 and 9 h were centrifuged and cell-free supernatant was added to the MRS nutrient broth inoculated with Bacillus stearothermophilus 794B. Inoculated cultures of this indicator organism were incubated overnight. It was found that the antibiotic activity was stable within 9h incubation interval and, thus, a resistance mechanism other than the direct inactivation of virginiamycin existed in virginiamycin-adapted wethers.