This study established the influence of dietary neomycin sulphate on the rate of hydrolysis of ochratoxin A (OA) by digesta from the intestine, and its effect on the excretion of OA and its hydrolyzed metabolite, alpha ochratoxin (O alpha), in the urine and feces of the rat. The first in vitro study demonstrated that digesta from the cecum and the large intestine were able to hydrolyze OA whereas digesta from the small intestine and stomach had very low hydrolytic activity against this substrate. Homogenates of the liver had no hydrolytic activity. The second in vivo study demonstrated that digesta from the large intestine and cecum of the neomycin treated rats was much less effective (P < 0.001) in promoting the hydrolysis of OA than digesta from the control rats. Neomycin when added directly to the in vitro system, however, did not affect the rate at which OA was hydrolyzed. In a third study, OA was administered in vivo to control and neomycin-treated rats. Rats fed the neomycin containing diet compared to those fed the control diet had a higher concentration (P < 0.005) of blood OA, and a greater cumulative excretion of OA plus O alpha over the entire 5 day collection period in the feces (P < 0.0001) and a corresponding decrease in the cumulative excretion of OA plus O alpha in the urine (P < 0.0001). Individually, there was a marked increase in cumulative fecal excretion of OA (P < 0.05) and a corresponding decrease in excretion of O alpha (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)