BACKGROUND The cytologic diagnosis of gastric adenocarcinoma often is difficult, and the role of gastric brushing in the detection of gastric malignancy is controversial. The purpose of this study was to identify the key cytologic criteria that are most useful for establishing a diagnosis of adenocarcinoma in gastric brushing specimens. METHODS One hundred gastric brushings were reviewed retrospectively. Fifty of the specimens were obtained from patients with histologically confirmed benign lesions. The other 50 specimens were obtained from patients with histologically confirmed gastric adenocarcinoma. All 100 brushing specimens were reviewed without knowledge of the histologic diagnosis. Each specimen was assessed for the presence or absence of 16 different cytologic features that have been identified in the published literature as being useful for separating benign conditions from malignancy. A multiple logistic linear regression analysis was performed to determine which combination of criteria was the most useful for diagnosing gastric adenocarcinoma. RESULTS Three key cytologic criteria were identified as being the most useful for diagnosing gastric adenocarcinoma: single atypical cells with intact cytoplasm, eccentric nuclei, and atypical naked nuclei. When at least 2 of these cytologic criteria were present, the sensitivity and specificity for detecting adenocarcinoma were 88% and 100%, respectively. Two minor cytologic criteria also were identified: nuclear hyperchromasia and nuclear molding. CONCLUSIONS Our statistical analysis demonstrates that gastric adenocarcinoma can be diagnosed with a high degree of accuracy using gastric brushing specimens when specific cytologic criteria are applied.