This study was performed to evaluate the utility of serum and cyst fluid analysis for enzymes (amylase and lipase) and tumor markers (carcinoembryonic antigen, CA 19-9, CA 125, and CA 72-4) in the differential diagnosis of cystic pancreatic lesions. Serum and cyst fluid were obtained from 48 patients with pancreatic cysts (21 pseudocysts, 14 mucinous cystic neoplasms, 6 ductal carcinomas, and 7 serous cystadenomas), observed between 1989 and 1994. Serum CA 19-9 levels were significantly higher in ductal carcinomas (all > 100 U/mL) and mucinous cystic neoplasms (P < 0.05). CA 72-4 cyst fluid levels were significantly higher in mucinous cystic tumors (P < 0.005), with 95% specificity and 80% sensitivity in detecting mucinous or malignant cysts. A combined assay of serum CA 19-9 and cyst fluid CA 72-4 correctly identified 19 of 20 (pre-) malignant lesions (95%), with only 1 false-positive result (3.6%). Cytology showed a sensitivity of 48% and specificity of 100%. Any pancreatic cyst with high serum CA 19-9 values, positive cytology, or high CA 72-4 in the fluid should be considered for resection.