Picornaviral proteinases are responsible for maturation cleavages of the viral polyprotein, but also catalyze the degradation of cellular targets. Using graphical visualization techniques and neural network algorithms, we have investigated the sequence specificity of the two proteinases 2Apro and 3Cpro. The cleavage of VP0 (giving rise to VP2 and VP4), which is carried out by a so-far unknown proteinase, was also examined. In combination with a novel surface exposure prediction algorithm, our neural network approach successfully distinguishes known cleavage sites from noncleavage sites and yields a more consistent definition of features common to these sites. The method is able to predict experimentally determined cleavage sites in cellular proteins. We present a list of mammalian and other proteins that are predicted to be possible targets for the viral proteinases. Whether these proteins are indeed cleaved awaits experimental verification. Additionally, we report several errors detected in the protein databases. A computer server for prediction of cleavage sites by picornaviral proteinases is publicly available at the e-mail address NetPicoRNA@cbs.dtu.dk or via WWW at http:@www.cbs.dtu.dk/services/NetPicoRNA/.