Bilitec 2000, a fiber-optic spectrophotometer, detects duodenogastric (DG) reflux in an ambulatory setting independent of pH by measuring bilirubin in bile. Studies were performed for independent validation and better understanding of limitations of this system. Bilitec and a conventional spectrophotometer were used to assess the effect of gastric acidity on the spectral absorbance of refluxed DG contents, employing bilirubin ditaurate (BDT) and human bile. Total bile acid concentrations correlated with Bilitec absorption. BDT and bile had hypsochromic shift (400 nm) in spectrophotometric maximum wavelength (lambda max; 450 nm) in an acidic environment (pH < 3.5), corresponding to an approximately 30% decrease in absorption measured by Bilitec. Bile acid concentrations correlated strongly with Bilitec absorption for all 63 samples (R = 0.82) with pH > 3.5 (n = 31, R = 0.81), but correlation was less strong for 32 samples with pH < 3.5 (R = 0.70). As currently designed, Bilitec adequately measures DG reflux but underestimates it by at least 30% in acidic medium (pH < 3.5). Design modifications are suggested for improving the sensitivity of this equipment.