Experiments were carried out at Uhříněves (Czech Republic) in 1996 and 1997 to evaluate the effects of avoparcin, bacitracin, flavomycin, tylosin and virginiamycin on in vitro fermentation of ovine ruminal contents and rabbit caecal contents. In incubations of the rumen contents supplied with starch, hemicellulose and pectin, all antibiotics at 20 μg/ml decreased production of fermentation gas and changed molar composition of fermentation products in favour of propionate. Flavomycin, tylosin and virginiamycin significantly decreased total production of volatile fatty acids (VFA). In incubations of the rabbit caecal contents, attempts to modify composition of microbial metabolites failed. Bacitracin was the only antibiotic which significantly increased the molar percentage of propionate and decreased acetate. This fermentation shift was accompanied by a decrease in total VFA production. The net production of propionate was thus similar in control and bacitracin-treated cultures. Rumen in vitro fermentation yielded more fermentation gas, methane and propionate than rabbit caecal fermentation of the same substrate. There was no apparent effect of antibiotics tested on methanogens in either microbial system, as the ratios of methane to VFA production were similar in control and treated cultures.