Microcapsule immunoassay (MCIA), a novel homogeneous Immunoassay system, involving protein-bearing liposome-encapsulated carboxyfluorescein as a release marker is reported. This system was applied for the determination of protein antigens such as fetritin, etc., in human serum samples by use of a sandwich assay technique. Liposomal lysis was observed in many samples, even though no second antibody was added to the reaction mixture. It was demonstrated that this phenomenon was related to the functional groups used to immobilize an antibody on liposomes. Stable liposomes in human sera could be prepared by incorporating bromoacetyl (BrAc) groups instead of the dithiopyridyl groups as used previously. A good correlation (y = 0.98, range = 10 -2000 pg 1-l) with data by radio immunoassay (RIA) was obtained in the ferritin measurement of 53 patients' sera by using these liposomes. To increase the sensitivity for the measurement of other tumor markers such as a-fetoprotein and carcinoembryonic antigen (CEA), the augmentation in the amounts of an antibody immobilired on the liposomal surface in MCIA was investigated. Based on the assumption that steric hindrance from liposomes would lit the immobilization of an antibody, the length of spacer between the antibody and the liposomal surface was optimized. The liposomes containing a BrAc-spacer-lipid (n = 5) showed a 50% increase in the antibody (Fab') immobilized, as compared with those without spacer molecules. CEA in the concentration of 0.1 pg 1-t in human sera could be detected, although correlation to RIA dropped to y -0.86 in 60 samples because of the increase in the nonspecific liposomal lysis based on human sera. The analyses of lipid monolayers using the Langmuir-Blodgett technique imply that the fluidity of lipid membranes may be related to instability of liposomes to human sera.