Voltage-sensitive dye (VSD) and calcium-sensitive dye (CaSD) imaging are important methods for studying the real-time spatiotemporal dynamics of cortical function in vivo. In this study we examine the cellular and network activity underlying the optical signals by combining these two imaging techniques in one preparation together with patch-clamp measurements. We first studied the cellular and biophysical properties of VSDs finding small, rapid fluorescence changes correlating linearly with membrane potential. CaSDs in individual neurons showed large fluorescence changes with a prolonged duration in response to action potentials, without prominent signaling of subthreshold events. At the network level, VSD and CaSD signals were differentially affected by pharmacological manipulations indicating that VSD signals correlated with postsynaptic subthreshold potentials whereas CaSDs reflected action potential firing. Consistent with this, the time-course of VSD responses followed neuronal membrane potential in vitro and in vivo. CaSD signals were spatially localized whilst the VSD signals spread across large cortical areas, reflecting narrow suprathreshold classical receptive fields in contrast to broad subthreshold receptive fields. Combined VSD and CaSD measurements can therefore be used to specify the temporal and spatial relationships between subthreshold and suprathreshold activity of the neocortex.