In brain slices, excitatory synaptic stimulation results typically in transient initial decreases in NAD(P)H fluorescence, followed by longer-lasting NAD(P)H increases that overshoot pre-stimulus NAD(P)H levels before returning slowly to baseline. We concluded recently that mitochondrial metabolism (rather than NADH generation by glycolysis) was responsible for the “overshoot” phase of responses evoked in murine hippocampal slices. The present study examined factors that may influence the amplitude of the overshoot phase, without necessarily directly influencing mitochondrial or glycolytic metabolism. The amplitudes of overshoots were influenced strongly by changes in pre-stimulus NAD(P)H fluorescence levels produced by a prior electrical stimulus. In contrast, these changes in pre-stimulus redox state had little effect on the amplitude of evoked initial NAD(P)H decreases. Resting NAD(P)H fluorescence levels differed significantly across sub-regions of each slice, however, this is not due to differences in resting redox state, and the relative amplitude of NAD(P)H overshoots were not different in different slice regions. Exposure to an A1 receptor agonist (CPA) reduced the amplitude of postsynaptic potentials, and preferentially reduced the amplitude of NAD(P)H overshoots, before initial oxidizing components of biphasic transients were reduced significantly. These results suggest that amplitudes of NAD(P)H overshoots may not be good quantitative measures of the intensity of a discrete stimulus, under some conditions where the stimulus is small relative to recent activity in the slice. Comparison of flavoprotein autofluorescence with NAD(P)H levels seems useful when making quantitative comparisons of responses from different regions of slices, where optical properties and ongoing activity may be substantially different. © 2007 Wiley-Liss, Inc.