This is for the first time that an attempt was made to enhance the performance of cross-flow ultrafiltration (CFUF) for the recovery and purification of lipopeptides by size-conditioning their micelles with Ca2+ ions and operating the system in diafiltration mode. In this study, polyether sulfone (PES) membranes with MWCOs 10, 30 and 50 kDa were evaluated for recovery and purification of lipopeptides produced by Bacillus sp. CFUF studies performed under batch mode showed poor recovery (68 +/- 2%) of F-2 Fraction comprising Fengycin isoforms, whereas F-1 fraction comprising a mixture of surfactin and iturin isoforms showed a moderate recovery of 83.5 +/- 2.4% in 50 kDa MWCO membrane. To improve the recovery, lipopeptides were conditioned with Ca2+ ions. The CFUF purification of this size-conditioned lipopeptide solution under diafiltration mode was carried out in 50 kDa PES membrane. Continuous irrigation of Ca2+ to lipopeptide solution under diafiltration mode of CFUF resulted in the increased recovery of lipopeptides, F1 (surfactin + iturin) fraction about 90.5 +/- 2.1% and F-2 about 95.3 +/- 2.0% Further, the colored impurities with MW range of 2334-3506 Da were effectively removed from the solution. The results of CFUF purification of Ca2+ conditioned lipopeptide solution under diafiltration mode indicate that this process can be readily scaled up to the commercial level, workable even with higher MWCO membranes. (c) 2013 Elsevier B.V. All rights reserved.