Some reactions of organic synthesis require to be performed in rather aggressive media, like organic solvents, that frequently impair enzyme operational stability to a considerable extent. We have studied the option of developing a reactivation strategy to increase biocatalyst lifespan under such conditions, under the hypothesis that organic solvent enzyme inactivation is a reversible process. Glyoxyl agarose immobilized penicillin G acylase and crosslinked enzyme aggregates of the enzyme were considered as biocatalysts performing in dioxane medium. Reactivation strategy consisted in re-incubation in aqueous medium Of the partly inactivated biocatalysts in organic medium, best conditions of reactivation being studied with respect to dioxane concentration and level of enzyme inactivation attained prior to reactivation. Best results were obtained with glyoxyl agarose immobilized penicillin G acylase at all levels of residual activity studied, with reactivations up to 50%; for the case of a biocatalyst inactivated down to 75% of its initial activity, full recovery of enzyme activity was obtained after reactivation. The potential of this strategy was evaluated in the thermodynamically controlled synthesis of deacetoxycephalosporin G in a sequential batch reactor operation, where a 20% increase in the cumulative productivity was obtained by including ail intermediate stage of reactivation after 50% inactivation. Biotechnol. Bioeng. 2009;103: 472-479. (C) 2009 Wiley Periodicals, Inc.