A flow injection liposome immunoanalytical system was developed using biotin as the model analyte and liposomal aequorin as the label. Aequorin is a photoprotein isolated from luminescent jellyfish (notably Aequorea victoria) and other marine organisms that emits visible light in the presence of a trace of Ca2+. Because of this characteristic, the aequorin complex has been used as an intracellular Ca2+ indicator. In this study, a bioluminescent label was designed by encapsulating aequorin inside the cavity of the liposome, whose outer surface was sensitized with the analyte of interest. The analyte-tagged liposomal aequorin was employed in the development of a heterogeneous bioluminescence immunoassay for the model analyte biotin. The proposed immunoassay was based on the competition between the model biotin and aequorin-encapsulating, biotin-tagged liposomes for a limited number of anti-biotin antibody-binding sites. The anti-biotin antibodies were immobilized via protein A in a capillary immunoreactor column, and 30% MeOH was used for the regeneration of antibody-binding sites after each measurement, which allowed the immunoreactor to be used for up to 50 sequential sample injections without any loss of reactivity. The calibration curve for biotin in Tris-buffered saline solution had a linear range of 1 x 10(-11)-1 x 10(-3) M. The detection limit of the assay was 50 pg (equivalent to 200-microL injection of 1 x 10(-9) M). This study demonstrates the procedures for the encapsulation of the photoprotein aequorin into the liposome, which can be used as a sensitive label in bioluminescence immunoassays for biotin or in other applications.