Lasalocid and monensin effectively reduced the lactate produced during in vitro fermentation of various sugars and ground grains with rumen fluid from either hay- or grain-fed cattle. The minimum effective dose was 6 micrograms/ml. Both lasalocid and monensin at 6 micrograms/ml reduced the fermentation rate of glucose, fructose, galactose, sucrose, lactate, mannose, ground corn, ground sorghum, and ground wheat, and raised the pH and substantially lowered lactate concentration compared with controls. Although both antibiotics decreased total lactate production, the proportion of D(-) lactate to L(+) lactate increased. Apparently both antibiotics were less inhibitory to the formation of D(-) than to L(+) lactate isomer. Lasalocid was more effective than monensin in inhibiting lactate production except when rumen fluid from grain-fed cattle was used in fermentation.