We have developed a liposome-based flow injection immunoassay (FIIA) system for quantitation of a clinical analyte, theophylline. With very minor changes in assay format, this procedure can also be used for the quantitation of anti-theophylline. Automated sequential analyses were performed at room temperature with picomole sensitivity and a day-to-day coefficient of variation of less than 5% for aqueous solutions. The system components include liposomes that contain fluorophores in their aqueous centers and an immobilized-antibody reactor column. The immunoreactor was regenerated hundreds of times over 3 months of continuous use with no measurable loss of antibody activity. The two assay formats studied produced distinct dynamic ranges for their respective analytes. The special advantages of using flow injection analysis for immunoassays and of using liposomes in FIIA are discussed.