It has been long known that the physiological concentrations of polyamines putrescine, spermidine and spermine are essential for cell growth. However, the role of endogenous polyamines in behavior function is poorly understood at present. This study investigated animals' behavioral performance and neurochemical changes in the hippocampus and prefrontal cortex following i.c.v. microinfusion of difluoromethylornithine (DFMO), a potent inhibitor of putrescine synthesis. Rats with low (25 microg) and high (50 microg) doses of DFMO spent significantly less time on the open arms and more time on the enclosed arms in the elevated plus maze relative to the saline controls, with no performance changes in the open field. The two DFMO groups were not impaired in the place and cued navigation, reversal training and probe tests in the water maze task. In the object recognition memory task, all three groups could detect the novel object, but rats in the high dose DFMO group spent significantly less time exploring displaced objects relative to the controls. DFMO treatment at both doses resulted in an 80%-90% reduction of putrescine level in the CA1, CA2/3 and dentate gyrus (DG) sub-regions of the hippocampus and the prefrontal cortex with minimal effects on the spermidine and spermine levels. Decreased spermidine/spermine molar ratio was found in DG and there were increased glutamate and GABA levels and their molar ratios in a region-specific manner in the DFMO groups. These results demonstrate that acute depletion of putrescine by DFMO produces anxiety-like behavior and impairs memory for the object displacement without affecting animals' locomotor and exploratory activity and spatial learning and memory. Multiple regression analysis data suggest the different roles of endogenous putrescine, spermidine and spermine on behavior function. The spermidine/spermine and glutamate/GABA ratios in hippocampal DG are strongly associated with anxiety-like behavior in the DFMO rats.