Alterations of monoamine metabolites in CSF and behavioral abnormalities were studied in rats with neonatal hippocampal lesions and controls. Lesions of the ventral hippocampus were produced bilaterally by ibotenic acid on postnatal day 7. Lesion-induced neurochemical alterations and behavioral impairments were examined concurrently when rats were 12 weeks old. CSF from the cisterna magna was sampled repeatedly from freely moving rats. The levels of free 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in CSF were determined. An exposure to a novel environment induced hyperexploratory behavior and elevated the level of free DOPAC in CSF in lesioned rats. Although a swim stress increased the levels of free DOPAC and 5-HIAA in CSF in both control and lesioned groups, rats with hippocampal lesions had a further elevation of free DOPAC in CSF and greater spontaneous activity relative to controls shortly after stress. Amphetamine (1.5 mg/kg, i.p.) induced hyperlocomotion in lesioned rats compared to controls. For the control group, the levels of the three monoamine metabolites in CSF were not significantly influenced by amphetamine. However, for the lesioned group, the level of DOPAC significantly decreased compared to preinjection of amphetamine. The results indicate that neonatal hippocampal lesion-induced impairments can be manifested by behavioral and neurochemical abnormalities. Alterations of monoamine metabolites in CSF may be determined quantitatively and used as indices for monitoring lesion-impaired monoaminergic function in the central nervous system.