Imaging of intrinsic optical signals has become an important tool in the neurosciences. To better understand processes underlying changes in intrinsic optical signals, we studied electrical stimulation at varying strengths in hippocampal slices of adult Wistar rats. Following serial stimulation we observed an increase in light transmittance in all tested slices. During antidromic stimulation at minimum stimulation strength the increase in light transmittance was 75 +/- 8% (P < 0.05), and during orthodromic minimum stimulation 19.6 +/- 5.6% (P < 0.001) in the stratum pyramidale of the CA1-region. During orthodromic stimulation no significant difference between submaximum, maximum and supramaximum stimulation was found, indicating saturation. In contrast, submaximum antidromic stimulation yielded 56.2 +/- 12% (P < 0.05) of maximum stimulation strength, indicating recruitment. In a further set of experiments serial stimulation was carried out under glial blockade with fluoroacetate (FAC) or blockage of mitochondrial function. Amplitude and slope of the intrinsic optical signal significantly decreased in the presence of FAC (amplitude: 36 +/- 6%, P < 0.01; slope: 37 +/- 11% as compared with baseline conditions, P < 0.05). This suggests a glial participation in signal generation. Rotenone, an inhibitor of mitochondrial complex I, yielded decreased amplitudes of the intrinsic optical signal (27 +/- 7% after 40 min, P < 0.01). Our data indicate that the intrinsic optical signal change reflects type and strength of neuronal activation and point to glia and mitochondria as important participants in signal generation.