Separation by solvent sublation has been used for the first time in an aqueous two-phase system, and a new concept, aqueous two-phase flotation (ATPF), is proposed. The new technique was used to separate and concentrate penicillin G from a fermentation broth. In an ATPF system of polyethylene glycol (PEG)/ammonium sulfate, the effects of varying solution pH, concentration of ammonium sulfate in aqueous solution, nitrogen flow rate, flotation time and initial volume of the PEG phase were investigated in detail, and the optimal conditions for ATPF were obtained. Under these optimal conditions, the separation efficiency of ATPF was more than 95%. Compared with liquid-liquid extraction, solvent sublation and aqueous two-phase extraction, ATPF is shown to be an effective way of separating and concentrating penicillin G from an aqueous phase, and also gives a reduced consumption of organic solvents. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.