When acetohydroxamic acid was incubated with washed suspensions of bovine rumen microorganisms the urease activity of the suspensions was depressed; activity could not be restored by the addition of divalent cations which, in the absence of acetohydroxamic acid, stimulated the urease activity of the cells. Acetohydroxamic acid was slowly degraded by the rumen microbiota. When the compound was incorporated into a non-selective medium for the enumeration of rumen bacteria it completely prevented visible colony development by some components of the inoculum and retarded the rate of multiplication of others. Acetohydroxamic acid inhibited the production of volatile fatty acids from added cellulose in strained rumen fluid and modified the molar proportions of acetate, propionate, and butyrate produced from the substrate; whereas in the absence of acetohydroxamic acid propionate production was favored at the expense of acetate; in its presence the acetate:propionate ratio remained constant. The effect of acetohydroxamic acid upon rumen microbial activities in vitro was therefore not limited to inhibition of rumen urease. It was impossible, however, to infer from the results obtained whether the potential value of the compound as a urease inhibitor in vivo would be diminished for this reason; this is because the influence of acetohydroxamic acid on the rumen microbiota in vivo is probably subject to modification by factors, such as the composition of the animal's diet, which were not investigated.