Summary For the evaluation of the effectiveness of phytase preparations as feed additive, in vitro properties like temperature optimum, temperature stability, pH optimum and pH profile or proteolytic stability are of utmost importance. Although at present all commercial phytase preparations authorized as feed additives in the EU are produced by recombinant filamentous fungi and have similar in vitro properties (acidic pH optimum, narrow pH range, low thermostability) the diversity of microbial phytases is great. Microbial sources for phytases span from fungi and yeasts to bacteria. Some of the naturally occurring phytases were identified to have high thermostability and a broad pH range (e.g. Aspergillus fumigatus phytase). The bacterial Bacillus phytases generally differ from other phytases, having a pH optimum from 7.0 to 8.0, being Ca2+ dependent and highly specific for phytate. Thermostability can considerably be increased by protein engineering. A so-called Consensus phytase encoded by a synthetic gene was found to be stable in aqueous solutions at 70 °C and in feed at pelleting temperatures of 80–90 °C. The rate and site of inactivation of feed enzymes in the digestive tract are determined by their susceptibility to proteolytic enzymes. Highest residual activities after incubation in the presence of pepsin or in supernatants of stomach digesta was observed for Escherichia coli and Consensus phytases, while the Bacillus phytase was found to be most resistant to pancreatin. Comparative studies on in vitro properties of enzymes intended for use as feed additives provide valuable information for prediction of in vivo effectiveness.