AbstractTheory suggest that networks of neurons may predict their input. Prediction may underlie most aspects of information processing and is believed to be involved in motor and cognitive control and decision-making. Retinal cells have been shown to be capable of predicting visual stimuli, and there is some evidence for prediction of input in the visual cortex and hippocampus. However, there is no proof that the ability to predict is a generic feature of neural networks. We investigated whether random in vitro neuronal networks can predict stimulation, and how prediction is related to short- and long-term memory. To answer these questions, we applied two different stimulation modalities. Focal electrical stimulation has been shown to induce long-term memory traces, whereas global optogenetic stimulation did not. We used mutual information to quantify how much activity recorded from these networks reduces the uncertainty of upcoming stimuli (prediction) or recent past stimuli (short-term memory). Cortical neural networks did predict future stimuli, with the majority of all predictive information provided by the immediate network response to the stimulus. Interestingly, prediction strongly depended on short-term memory of recent sensory inputs during focal as well as global stimulation. However, prediction required less short-term memory during focal stimulation. Furthermore, the dependency on short-term memory decreased during 20 h of focal stimulation, when long-term connectivity changes were induced. These changes are fundamental for long-term memory formation, suggesting that besides short-term memory the formation of long-term memory traces may play a role in efficient prediction.