The experiments on reactive extraction of gentamicins C-1, C-1a, C-2 and C-2a with D2EHPA dissolved in dichloromethane indicated the possibility to selectively separate the gentamicin C-1 from the mixture obtained by biosynthesis. The selectivity of its extraction is positively influenced by increasing the pH-value of aqueous solution and by using a D2EHPA concentration in organic phase near the value needed for an equimolecular ratio between the extractant and this gentamicin. The maximum value of the selectivity factor has been reached at pH 8 and 10 g(l-1) D2EHPA. Therefore, the raffinate will be enriched in gentarnicins C-1a C-2 and C-2a, their overall ratio increasing from 68% in the biosynthetic product to 84.6%. The second step of the experiments consists on the recovery of gentamicins C-1, C-1a, C-2 and C-2a from the extracts previously obtained by reextraction with sulfuric acid solutions. Thus, it is possible to re-extract mainly the most active gentamicins, namely C-1a, C-2 and C-2a. The selectivity of their separation is positively influenced by using the sulfuric acid concentrations near to the values stoichiometric needed for reacting only with gentarnicins C-1a, C-2 and C-2a. The mixture resulted by re-extraction contains 92.6% gentarnicins C-1a, C-2 and C-2a. By bringing together the aqueous solutions obtained by reactive extraction and re-extraction, the ratio of the active gentarnicins becomes 89%, their loss reaching 0.3% related to the initial biosynthetic product. (C) 2007 Elsevier B.V. All rights reserved.