Alpha fetoprotein (AFP) has been implicated in the development of hepatocellular carcinoma and is considered to be a diagnostic and prognostic tumor marker. Because elevated expression of AFP is associated with many characteristics of hepatocellular carcinoma tissues, we hypothesized that multiple proteins may function in a coordinated manner with AFP. To identify such proteins, we performed global protein expression analysis, namely a proteomic study. The protein expression profiles of 9 AFP-producing liver cancer cell lines (JHH-5, HuH-1, PLC/PRL/5, Hep3B, HT-17, JHH-7, HuH-7, HepG2, Li-7) and 7 nonproducing liver cancer cell lines (HLE, JHH-6, Sk-Hep-1, JHH-4, HLF, RBE, SSP-25) were generated by fluorescence 2-dimensional difference gel electrophoresis. In fluorescence 2-dimensional difference gel electrophoresis, proteins are labeled with fluorescent dyes before electrophoresis for more accurate quantitative expression analysis. We identified 11 protein spots that distinguished AFP-producing cell lines from nonproducing cell lines by multivariate studies. The spots showed consistent alterations in amount in AFP-producing cell lines (6 up-regulated and 5 down-regulated). An additional 5 liver cancer cell lines (KIM-1, KYN-2, KYN-3, PH5-CH, PH5-T) also were correctly grouped with respect to their AFP production on the basis of the intensity of the 11 protein spots. The proteins corresponding to the 11 selected spots were identified by mass spectrometry and were categorized into 4 groups based on their known role in apoptosis, glucose metabolism, cytoskeletal organization, or translation. In conclusion, we found a novel association of AFP with other proteins. Their interaction should provide insight into the biology of AFP-producing hepatocellular carcinoma cells. (HEPATOLOGY 2004;40:609 -617.