Pancreatic cystic neoplasms comprise a pathologically heterogeneous group with many shared clinical features. We assessed the reliability of cyst fluid analysis for the differential diagnosis of pancreatic cysts. Cyst fluid was obtained by fine-needle aspiration from 78 pancreatic cysts. The lesions studied consisted of 17 mucinous cystic tumors (MCTs), 13 serous cystadenomas (SCAs), 5 solid pseudopapillary tumors (SPTs), 8 intraductal papillary mucinous tumors (IPMTs), 6 ductal adenocarcinomas (ACAs) with cystic degeneration, and 29 pseudocysts (PCs). Epithelial cells were observed in 27 (81%) of 33 successful aspirates of cystic neoplasms. Cytologic diagnosis was possible in 5 (31%) out of 16 MCTs. Mucicarmine staining was positive in five out of nine MCTs, one out of one ACA, and one out of two IPMTs, but in none of the SCAs, SPTs, or PCs. Cyst fluid carcinoembryonic antigen (CEA) levels of more than 467 ng/mL had a 87% sensitivity and a 98% specificity for detecting MCTs, and amylase levels of more than 479 U/L had a 73% sensitivity and a 90% specificity for detecting PCs. In conclusion, cyst fluid analysis for cytology, mucin staining, CEA, and amylase levels are useful in the differential diagnosis of pancreatic cysts. Diagn. Cytopathol. 2004;31:100–105. © 2004 Wiley-Liss, Inc.