The effect of intraseptal injection of the cholinergic immunotoxin 192-IgG-saporin on behavior in the elevated plus maze was investigated. A 5-min test-retest paradigm, with minute-by-minute analysis of the first session, was used to evaluate both anxiety and memory in this task. Biochemical analyses revealed a decrease in acetylcholinesterase (AChE) activity in the hippocampus (HPC), septum, and frontal cortex of animals injected with IgG-192 saporin (237.5 ng) when compared with controls. No statistical differences were found between groups in terms of behaviors associated with locomotor activity, conventional measures of anxiety, or ethological behaviors during either session 1 or 2. During test session 2 the controls exhibited decreased exploratory activity and increased indices of anxiety. In contrast, the saporin-treated rats did not exhibit these experience-dependent behavioral changes from session 1 to 2. The minute-by-minute analysis showed a significant decrease in exploratory as well in anxiety associated behaviors during the first session for the control group, but not for the saporin-treated group. These results suggest that the cholinergic innervation of the HPC, the frontal cortex, or both forebrain structures, modulate the initiation of exploratory activity which, results in the acquisition and retention of spatial information, but does not affect the expression of anxiety in the elevated plus-maze.