Carcinoma cuniculatum, a unique variant of well-differentiated squamous cell carcinoma, has been only rarely reported in the esophagus. The present study was undertaken to determine if a previously observed common histologic pattern for carcinoma cuniculatum is diagnostically useful in esophageal mucosal biopsy specimens. Thirty-five esophageal mucosal biopsies obtained from 25 procedures in 11 patients with a resection-proven diagnosis of carcinoma cuniculatum were compared with 92 esophageal biopsies from 69 patients with benign diagnoses. All biopsies were assessed for the presence of hyperkeratosis, acanthosis, dyskeratosis, deep keratinization, intraepithelial neutrophils, neutrophilic microabscess, focal cytologic atypia, koilocyte-like cells, and keratin-filled cyst/burrows. Each feature, if present, was given one point, and the final histologic score was calculated for each biopsy by summing the points. The mean histologic score was 6.66 (s.d. 1.88) in biopsies from carcinoma cuniculatum vs a mean score of 1.93 (s.d. 1.75) for biopsies with benign diagnoses (P<0.0001). Using a cutoff value of 7 for carcinoma cuniculatum, 57% of biopsies (20/35) from 64% esophagogastroduodenoscopy procedures (16/25) in 91% patients (10/11) would be diagnostic, in comparison to the initial diagnostic rates of carcinoma of 9, 12, and 27%, respectively (P<0.0001 for all). None of the 92 benign biopsies showed a score of ≥7. Our results demonstrate that a semiquantitative histologic evaluation of mucosal biopsies taken from an esophageal mass greatly improves the diagnostic sensitivity from patients with carcinoma cuniculatum with 100% specificity. Larger studies are necessary to confirm the current findings.