O-Hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) is a chiral organophosphorus compound that undergoes enzymatic hydrolysis in the rat and hen. Studies of the stereospecificity of its biodegradation are necessary to establish HDCP toxicity. To this effect, methods have been developed for the analysis of the HDCP stereoisomers by gas chromatography (GC) and high-performance liquid chromatography (HPLC). The best resolution and analysis were obtained by HPLC with UV detection, a OA-4100 Techocel chiral column and the mobile phase: hexane-1,2-dichloroethane-ethanol (92:5:3, v/v/v). The detection limit was 25 microM for HDCP and 5 microM for one of its hydrolytic products: 2,5-dichlorophenol (DCP). The method was reproducible intra o inter die. Moreover, a method is described for the liquid extraction of HDCP and DCP with 1,2-dichloroethane in biological samples, with a yield of (80.3 +/- 9.7)% (n = 10, S.D.) for HDCP and (84.1 +/- 10.0)% (n = 10, S.D.) for DCP. The method is compared with the solid-phase extraction technique with C18 sorbent. The hydrolysis of HDCP by hen plasma is studied.