ABSTRACT: Predicting protein subcellular localization is an important and difficult problem, particularly when query proteins may have the multiplex character, i.e., simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular location predictor can only be used to deal with the single-location or "singleplex" proteins. Actually, multiple-location or "multiplex" proteins should not be ignored because they usually posses some unique biological functions worthy of our special notice. By introducing the "multi-labeled learning" and "accumulation-layer scale", a new predictor, called iLoc-Euk, has been developed that can be used to deal with the systems containing both singleplex and multiplex proteins. As a demonstration, the jackknife cross-validation was performed with iLoc-Euk on a benchmark dataset of eukaryotic proteins classified into the following 22 location sites: (1) acrosome, (2) cell membrane, (3) cell wall, (4) centriole, (5) chloroplast, (6) cyanelle, (7) cytoplasm, (8) cytoskeleton, (9) endoplasmic reticulum, (10) endosome, (11) extracellular, (12) Golgi apparatus, (13) hydrogenosome, (14) lysosome, (15) melanosome, (16) microsome (17) mitochondrion, (18) nucleus, (19) peroxisome, (20) spindle pole body, (21) synapse, and (22) vacuole, where none of proteins included has ?25% pairwise sequence identity to any other in a same subset. The overall success rate thus obtained by iLoc-Euk was 79%, which is significantly higher than that by any of the existing predictors that also have the capacity to deal with such a complicated and stringent system. As a user-friendly web-server, iLoc-Euk is freely accessible to the public at the web-site http://icpr.jci.edu.cn/bioinfo/iLoc-Euk. It is anticipated that iLoc-Euk may become a useful bioinformatics tool for Molecular Cell Biology, Proteomics, System Biology, and Drug Development Also, its novel approach will further stimulate the development of predicting other protein attributes.