The electrochemical immobilization of lactate oxidase in a poly-o-phenylenediamine film permits the one-step and all-chemical construction of a lactate amperometric biosensor. The sensor was prepared in situ i.e. in the flow injection analysis (FIA) system by simply injecting a plug of a solution containing the monomer and the enzyme. At a flow rate of 50 microL/min linearity was observed up to 0.2 mM lactate and detection limits of about 2 microM could be easily achieved. Faradaic interferences caused by ascorbate, urate, cysteine and acetaminophen were sufficiently minimized to permit lactate determination in diluted serum by FIA. Results obtained by FIA-amperometric detection compared well (according to a proper t-test at a 95% confidence level) with those obtained by a standard enzymatic colorimetric assay. At a flow rate of 1 ml/min a sample throughput higher than 70 sample h-1 was achieved. After one week of continuous use in the FIA system a 75% decrease in biosensor sensitivity was observed.