To characterize the physiological condition of human epileptic neocortex, we employed flavoprotein fluorescence imaging (FFI), an optical imaging method which detects intrinsic signals accompanying neural activation, and immunohistologically studied human cortical specimens. The experimented materials were cortical tissues surrounding various intracerebral lesions obtained from 5 patients with epilepsy (epileptic patients: EPs) and 5 without epilepsy (non-epileptic patients: NEPs). These tissues were immersed in oxygenated artificial cerebrospinal fluid immediately after removal in the operating room. Signal changes of FFI in the cortical layers subjected to electrical stimulation were observed under bicuculline methiodide perfusion. Immunohistological staining for parvalbumin (PV), calbindin, and calretinin were performed on the same specimens to evaluate expressions of calcium-binding protein positive cells. The FFI study showed the characteristic cortical propagation pattern of elicited activities horizontally along the cortical layers in EPs but not in NEPs. The propagated area with more than 0.5% signal changes was significantly larger in EPs than in NEPs (p=0.008). Only the expression of PV positive neurons was significantly lower in EPs than in NEPs (p=0.006). The propagated area on FFI and the decrease in PV positive neurons correlated significantly (R=-0.78, p=0.04). The present study visualized the unique horizontal propagation of signal changes on FFI and demonstrated a correlation of this propagation with immunohistological decreases in PV positive neurons in human epileptic cortex. Further investigations may elucidate the mechanism of hyper-excitability and hyper-synchronization in epileptic cortical tissue itself. © 2012 Elsevier B.V.