Immuno-supported liquid membrane (immuno-SLM) extraction is a new technique that makes use of antibody (Ab)-antigen interactions as the "extraction force" to drive the mass transfer in a selective way. In immuno-SLM, anti-analyte (Ag) Abs are introduced into the acceptor phase of the SLM unit to trap the Ag that passes from the flowing donor through the SLM into the stagnant acceptor. The amount of immuno-extracted analyte (AbAg) is quantified by connecting the immuno-SLM unit on-line with a non-competitive heterogeneous fluorescence flow immunoassay (FFIA) that makes use of a fluorescein-labeled analyte tracer that titrates the residual excess of Ab present in the acceptor. A restricted access (RA) column is used for the separation of the two tracer fractions (Ag* and AbAg*) formed, and the eluted AbAg* fraction is measured downstream by a fluorescence detector. Factors influencing the optimum immuno-SLM extraction parameters, i.e., donor flow rate, extraction time and type of Ab, were investigated for immuno extraction of the model analyte atrazine. Immuno-SLM coupled to FFIA (immuno-SLM-FFIA) and FFIA alone were compared in terms of the assay sensitivities obtained and the sample matrix influence. The concentration at the mid-point of the calibration curve (IC(50)) was 16.0+/-1.4 and 36+/-16 microg/l, the limit of detection (LOD) was 2.0+/-1.1 and 20+/-10 microg/l, and the dynamic range was 2-100 and 20-500 microg/l atrazine for immuno-SLM-FFIA and FFIA, respectively. The matrix influence on the FFIA was significant in orange juice and surface water, whereas the influence was minor for immuno-SLM-FFIA with recoveries between 104% and 115% for 5 microg/l atrazine in tap water, orange juice and river water.