Mixed ruminal bacteria (n = 4) were incubated in anaerobic media for 24 h in vitro with either hay, corn meal, protein hydrolyzate or hydrogen gas as the substrate. The ionophore monensin and the polypeptide antibiotic bacitracin were added to the incubation flasks at concentrations ranging from 0 to 10 or 40 mg/liter. As was expected, monensin decreased methane production, increased the ratio of propionate to acetate and decreased the deamination of amino acids. Monensin had little effect on methane production, however, if hydrogen gas was the fermentation substrate. Bacitracin, another gram-positive antibiotic with a distinctly different cellular target, was somewhat less potent than monensin, but it produced strikingly similar responses. This similarity of fermentation patterns suggested that monensin action in the rumen is probably due to its activity as a gram-positive antibiotic, and that any gram-positive antibiotic not suppressed by resistance may produce fermentation effects similar to those of monensin. The cellular action of monensin as an ionophore in membranes is probably little more than a means of inhibiting sensitive species. Many gram-positive antibiotics have little affect on ciliate protozoa or coccidia.