MIB-1, a proliferation marker may be useful in the assessment of esophageal biopsy specimens for gastroesophageal reflux disease (GERD). Forty-five hematoxylin and eosin-stained esophageal biopsy specimens were histologically assessed for basal zone height, papillary length, and inflammatory cell infiltrate and classified as 10 normal and 35 esophagitis. The percentage of MIB-1-positive area (MIB-1% area) was measured on immunostained sections using image analysis (CAS 200) in the basal half of well-oriented areas and adjacent to five cross-sectioned papillae (c-pap) in poorly oriented areas. The cell layer of the MIB-1-positive cell furthest from the basal layer of the c-pap was also noted. MIB-1% area was significantly greater in both well- and poorly oriented areas of esophagitis biopsy specimens compared with normal biopsy specimens. MIB-1 positivity in the basal half and c-pap were correlated (r = 0.43, p = 0.017). MIB-1 expression correlated with basal zone height and eosinophil infiltrate (r = 0.61, p < 0.001; r = 0,32, p = 0.03, respectively). The cell layer with positive cells furthest from c-pap in normal and esophagitis biopsy specimens was two and six layers, respectively. Using 31% as a threshold to detect abnormal findings, the MIB-1 sensitivity/specificity and positive predictive value in the basal half and c-pap were 86, 70, 91% and 80, 80, 94%, respectively. In summary, MIB-1 staining correlates with basal zone hyperplasia and eosinophil infiltrate seen in GERD. MIB-1 staining can be assessed both in well- and poorly oriented areas as MIB-1% areas. Alternatively simply finding MIB-1 positive cells more than three cell layers from the basal layer is abnormal and consistent with GERD.