A simple, fast, and fully automated FIA-SPE method with UV detection for the preconcentration and determination of the investigated penicillins has been developed. This paper provides adequate procedure for the preconcentration and determination of the studied compounds in pharmaceuticals and milk samples. Penicillins (penicillin G, amoxicillin, and ampicillin) are extracted in a mincolumn packed with methylene blue grafted polyurethane foam (MBGPUF) material. The antibiotics are eluted by hydrochloric acid solution to the flow cell of UV-vis spectrophotometer at 230 nm. The analytes are preconcentrated on the sorbent at pH 8.0-9.5 and sample flow rate 3.0 mL/min. Elution was performed with 200 mu L 0.2 mol L-1 hydrochloric acid at 2 mL min(-1). Sample throughput is 12 h(-1) at 120 s preconcentration time. High selectivity of the sorbent for the analytes was achieved at the specified pH range. The enrichment factors achieved are 14, 16, and 11 with 3 sigma detection limits of 12, 15, and 19 ng mL(-1) for penicillin G, amoxicillin and ampicillin, respectively. The method was successfully applied to the determination of these antibiotics in pharmaceutical control and contaminated milk samples with RSD <= 8.8%. (C) 2010 Elsevier B.V. All rights reserved.