A fiber-optic enzyme biosensor for the direct measurement of organophosphate nerve agents was developed. The basic element of this biosensor is organophosphorus hydrolase immobilized on a nylon membrane and attached to the common end of a bifurcated optical fiber bundle. The enzyme catalyzes the hydrolysis of organophosphate compounds to form stoichiometric amounts of chromophoric products that absorb light at specific wavelengths. The back-scattered radiation of the specific incident radiation was measured using a photomultiplier detector and correlated to the organophosphate concentration. The effects of buffer pH, temperature, and the units of enzyme immobilized on the steady-state and kinetic response of the biosensor were investigated to optimize the operating conditions for the fiber-optic enzyme biosensor. These conditions were then used to measure parathion, paraoxon, and coumaphos selectively without interference from carbamates and triazines. Concentrations as low as 2 mu M can be measured in less than 2 min using the kinetic response. When stored in buffer at 4 degrees C the biosensor shows long-term stability.