Ambulatory 24-hour esophageal pH monitoring is the gold standard examination to assess esophageal acid exposure. Gender-related variation is a well-recognized physiologic phenomenon in health and disease. To date, limited gender-specific 24-hour esophageal pH monitoring data are available. The aim of this study was to obtain values of esophageal pH monitoring in males and females without reflux symptoms or gastroesophageal reflux disease (GERD) to determine if gender variation exists in esophageal acid exposure among individuals without these factors. Twenty-four-hour dual esophageal pH monitoring was performed in male and female volunteers without reflux symptoms or GERD. Values for total number of reflux episodes, episodes longer than 5 minutes, total reflux time in minutes, % time with pH below 4, and longest reflux episode in the proximal/distal esophagus were obtained and recorded for both groups. The distal channel was placed 5 cm and proximal channel 15 cm above the manometrically determined lower esophageal sphincter. Means were compared using an independent sample t-test. Sixty-seven males and 69 females were enrolled. All subjects completed esophageal 24-hour pH monitoring without difficulty. There was no age or body mass difference between groups. Females had significantly fewer reflux episodes at both esophageal measuring sites and, significantly less total reflux time and % time with pH below 4 in the distal esophagus than males. All other parameters were similar. Significant gender-related differences exist in esophageal acid exposure, especially in the distal esophagus in individuals without reflux symptoms or GERD. These differences underscore the need for gender-specific reference values for 24-hour pH monitoring, allowing for an accurate evaluation of esophageal acid exposure in symptomatic patients.