When the standard approach to predict protein function by sequence homology fails, other alternative methods can be used that require only the amino acid sequence for predicting function. One such approach uses machine learning to predict protein function directly from amino acid sequence features. However, there are two issues to consider before successful functional prediction can take place: identifying discriminatory features, and overcoming the challenge of a large imbalance in the training data. We show that by applying feature subset selection followed by undersampling of the majority class, significantly better support vector machine (SVM) classifiers are generated compared with standard machine learning approaches. As well as revealing that the features selected could have the potential to advance our understanding of the relationship between sequence and function, we also show that undersampling to produce fully balanced data significantly improves performance. The best discriminating ability is achieved using SVMs together with feature selection and full undersampling; this approach strongly outperforms other competitive learning algorithms. We conclude that this combined approach can generate powerful machine learning classifiers for predicting protein function directly from sequence. © 2005 Adis Data Information BV. All rights reserved.