Diarrhea in neonates is often complicated by metabolic acidosis. We used blood gas analysis and HPLC to determine whether bacterial fermentation might contribute to acidosis in diarrheic calves. Diarrheic calves (n = 21) had significantly lower pH, PCO(2), HCO(3)(-) and a higher anion gap than healthy calves (n = 21). Serum concentrations (mean +/- SD, mmol/L) of DL-, L- and D-lactate were also significantly higher in diarrheic (8.9 +/- 5.1, 4.1 +/- 3.4 and 5.2 +/- 5.7) than in healthy calves (1.7 +/- 1.2, 2.0 +/- 1.1 and too low to quantify). D- and L-lactate accounted for 64% anion gap increase in diarrheic calves. Fecal D- and L-lactate concentrations were also significantly higher in diarrheic calves (9.4 +/- 3.0 and 11.9 +/- 2.7 mmol/L) than healthy calves (1.1 +/- 0.1 and 1.6 +/- 0.1 mmol/L). The elevated concentrations of serum and fecal D-lactate suggest gut bacterial fermentation contributes to the development of acidosis in diarrhea.