Using unanaesthetized and free-moving mature female cats, we examined changes of cortisol levels in 24-hr urine accompanying emotional behaviour such as restlessness elicited by electrical stimulation of the anteromedial hypothalamus(AMH). Urine was collected every 24 hr for 9 consecutive days and stimulation was applied for only the middle 3 days with chronically implanted electrodes placed in the AMH. Cat urine was hydrolysed with β-glucuronidase and sulphatase; the levels of free cortisol, glucuronide and sulphate were determined by fluorescence polarization immunoassay. The mean (fSD) of the total cortisol level calculated in terms of the creatinine level in the 3 days before stimulation was 13.04 ± 4.89 ng/mg creatinine; the mean for the 3 days during stimulation was 19.06 ± 7.56 ng/mg creatinine; and the mean for the 3 days after stimulation was 13.97 ± 4.10 ng/mg creatinine. The cortisol level during stimulation showed a significant increase compared with that before stimulation (p < 0.05) and almost recovered to the prestimulation level 3 days after stimulation. These results indicate that the emotional behaviour such as restlessness induced by AMH stimulation in cats may have some relation to emotional stress.