A flow injection (FI) enzymatic system, based on the use of immobilized salicylate hydroxylase in glass beads, was developed for the determination of salicylate. Salicylate hydroxylase and nicotinamide adenine dinucleotide (NADH) are used to convert salicylate to catechol. The reaction of catechol with 4-aminophenol at high pH yields a colored product which is detected spectrophotometrically at 565 nm. Ten samples of human serum containing from 5.0 x 10(-4) to 5.0 x 10(-3) mol/L added salicylate were analyzed and the recovery was determined. Eight additional serum samples containing salicylate were analyzed by the Trinder test and the proposed method. The results obtained with the 2 methods showed good agreement by the statistical Student's t-test. The relative precision of the method is about 3.4% (RSD of the mean recovery). Considering the lowest concentration analyzed, the quantitative limit of detection is about 0.2 x 10(-5) mol/L (3 x SD). The volume of the sample used was 150 microL. The proposed method was also used to analyze medicines containing acetylsalicylic acid. The results were statistically compared with those obtained through the U.S. Pharmacopoeia procedure and showed excellent agreement.