We present a new program for predicting protein subcellular localization from amino acid sequence. WoLF PSORT is a major update to the PSORTII program, based on new sequence data and incorporating new features with a feature selection procedure. Following SWISS-PROT, we divided eukaryotes into three groups: fungi, plant, and animal. For the 2113 fungi proteins divided into 14 categories; we found that, combined with BLAST, WoLF PSORT yields a cross-validated accuracy of 83%, eliminating about1/3 of the errors made when using BLAST alone. For 12771 animal proteins a combined accuracy of 95.6% is obtained, eliminating 1/4 of BLAST alone errors, and for 2333 plant proteins the accuracy can be improved to 86% from 84%.