Whole organism-based small-molecule screens have proven powerful in identifying novel therapeutic chemicals, yet this approach has not been exploited to identify new cognitive enhancers. Here we present an automated high-throughput system for measuring nonassociative learning behaviors in larval zebrafish. Using this system, we report that spaced training blocks of repetitive visual stimuli elicit protein synthesis-dependent long-term habituation in larval zebrafish, lasting up to 24 h. Moreover, repetitive acoustic stimulation induces robust short-term habituation that can be modulated by stimulation frequency and instantaneously dishabituated through cross-modal stimulation. To characterize the neurochemical pathways underlying short-term habituation, we screened 1,760 bioactive compounds with known targets. Although we found extensive functional conservation of short-term learning between larval zebrafish and mammalian models, we also discovered several compounds with previously unknown roles in learning. These compounds included a myristic acid analog known to interact with Src family kinases and an inhibitor of cyclin dependent kinase 2, demonstrating that high-throughput chemical screens combined with high-resolution behavioral assays provide a powerful approach for the discovery of novel cognitive modulators.acoustic startle response | sensorimotor gating A ll organisms, from protozoa to humans, use nonassociative habituation learning as a means to update behavioral responses to sensory input based on recent stimulation history (1). Considered a simple form of learning, habituation reflects a suppressed behavioral response to repeated inconsequential stimulation and serves as a mechanism by which the nervous system filters irrelevant stimuli. Defective habituation not only is indicative of a learning deficit, but also is prevalent in neuropsychiatric conditions, such as schizophrenia, attention deficit hyperactivity disorder, posttraumatic stress disorder, and drug addiction (2-5). Numerous assays have shown that the parameters and rules for habituation learning are similar across phyla (6-9), suggesting conservation of the underlying molecular mechanisms. For example, training session design and stimulation frequency is predictive of the time scale of memory retention. Massed exposure to repeated stimulation at short interstimulus intervals (ISIs) elicits an acquisition of learned information with short-term recall memory, indicated by a change in behavior that does not persist for long after the repeated stimulation is terminated. In contrast, a distributed training protocol with multiple training sessions consisting of stimulation at longer ISIs and rest periods between training blocks induces the acquisition and storage of learned behavior that is capable of being recalled for a more extended period. Thus, intrasession, short-term habituation represents working memory, whereas intersession, long-term habituation includes the storage and retrieval of memory.Existing strategies to measure vertebrate lea...