Bioconversion of penicillin G in PEG 20000/dextran T 70 aqueous two-phase systems was achieved using the recombinant Escherichia coli A56 (ppA22) with an intracellular penicillin acylase as catalyst. The best conversion conditions were attained for: 7% (w/v) substrate (penicillin G), enzyme activity in bottom phase 52 U ml−1, pH 7.8, temperature 37 °C, reaction time 40 min. Five repeated batches could be performed in these conditions. Conversions ratios between 0.9–0.99 mol of 6-aminopenicillanic acid (6-APA) per mol of penicillin G, were obtained and volumetric productivity was 3.6–4.6 μmol min−1 ml−1. In addition the product 6-APA could be directly crystallized from the top phase with a purity of 96%.