Acetohydroxamic acid at a concentration of 1 × 10−6 M, 6 × 10−5 M, and 1 × 10−3 M inhibited urease of intact rumen microbes in vitro by 11%, 50%, and 74%. Inhibition of rumen urease by the acid reached equilibrium state, unlike jack bean urease. Inhibition was maximum over a broad range of pH (8 to 10) and it did not resemble the pH activity profile of rumen urease. Sulfhydryl compounds did not reverse the inhibition; however, addition of these compounds prior to acetohydroxamic acid addition prevented inhibition. The nature of inhibition was noncompetitive with inhibitor constant 4.8 × 10−5 M. Acetohydroxamic acid at a concentration that produced 50% urease inhibition did not affect rumen cellulase and proteolytic enzymes in vitro. The complex of acetohydroxamic acid-rumen urease is dissociable on dialysis.