Continued usage of cocaine is determined by genetic, conditioned and homeostatic factors, while it is reinforced by drug-induced reward and the emotionally negative state of drug withdrawal, which includes anxiety. The molecular mechanisms of these long-term behavioral and physiological alterations have yet to be fully elucidated. Here we demonstrate that in zebrafish, a wide range of non-anesthetic cocaine doses, 0.015-15 muM, does not result in acute alterations in locomotor activity, in spite of the high brain cocaine levels induced (7-120 pg/microg protein). Conversely, cocaine withdrawal causes hyperactivity associated with stereotypy. The behavioral hyperactivity is progressively increased during the initial period of withdrawal (24-72 h) and is maintained for at least 5 days. Such effect of cocaine withdrawal is aggravated by environmental stimulation and attenuated in the home environment. Administration of cocaine (1.5 microM) or a non-sedative dose of diazepam (5 microM, immersion) acutely counteracts withdrawal-associated hyperactivity and stereotypy in zebrafish, with the magnitude of these effects positively correlating with the degree of prior increase in basal activity. Administration of an anxiogenic benzodiazepine inverse agonist, FG-7142, results in zebrafish behavior similar to that observed during cocaine withdrawal. Together, the results suggest that cocaine withdrawal produces long-lasting behavioral effects in zebrafish which are consistent with an anxiety-like state. Thus, zebrafish, a powerful model for the study of vertebrate genetics, could provide insights into the molecular mechanisms of drug withdrawal.