Factors that control the performance of a reversible immunosensor with an analyte (progesterone)-enzyme (horseradish peroxidase) conjugate as signal generator have been investigated. The conjugate is used in conjunction with two antibodies, which are specific to progesterone and to horseradish peroxidase, immobilized on two spatially separated polypropylene mesh discs. The conjugate and two antibodies are confined to an internal compartment of a microdialyzer by a semi-permeable membrane. The small analyte from an external medium permeates across the membrane into the internal compartment where the analyte concentration determines the relative amounts of the bound conjugate on the two solid surfaces. By measuring two signals from the conjugate bound at two separate sites, we experimentally obtained time-response curves to a concentration pulse of the external analyte. A mathematical (kinetic) model describing the sensor system was developed and used for the determination of rate-limiting factors. In semicontinuous monitoring of the analyte concentrations, operation of the immunosensor with the enzyme conjugate as signal generator required special attention to (a) enzyme stability, (b) analyte permeation (dependence on medium components), and (c) kinetics related to the different accessibility to the same antibody of the small analyte (to be measured) vs. the larger counterpart on the enzyme conjugate (for signal generation).