Recent studies on the use of volatile electrolytes such as CO2 in protein precipitation showed that these agents are a promising alternative to the conventional acids. This use of volatile electrolytes prevents protein denaturation due to local pH extremes, and saline effluent generation is greatly reduced, as the volatile electrolyte may be separated and recovered from solution just by pressure release. In this work, insulin was successfully crystallized in the presence of zinc using CO2 as acidifying agent. The crystals obtained were rhombohedral, a common shape for porcine insulin crystals that contain zinc in their structure, and their average size varied with the mixing applied. (C) 2009 Elsevier B.V. All rights reserved