Polarographic fluoroimmunoassay was developed for identification and estimation of phenobarbital in urine. The study was carried out using TDx-analyzer "Abbott" (USA) under conditions of automatic service by modified program N 21 intended for analysis of kanamycin. In the procedure developed rabbit antiserum against phenobarbital was used at the initial dilution 1:2 and the tracer 5-ethyl-5-(4'-fluoroscein thiocarbamyl aminophenyl)barbituric acid was applied at the initial concentration of 60 nmol/l. The minimal content of phenobarbital evaluated in urine was as small as 0.09 microgram/ml and it was measured in the limits of 0.5 microgram/ml. Specificity of the analysis and its reproducibility were studied. The results of the polarographic fluoroimmunoassay correlated highly to the data of EMIT-st-assay and HPLC.