Gap junctions are essential for glial cell function and have been increasingly implicated in multiple sclerosis (MS). Because increasing cortical abnormalities correlate with disease progression and cognitive dysfunction, we examined the expression of oligodendrocytic connexin32 (Cx32) and Cx47 and their astrocytic partners Cx30 and Cx43 in cortical lesions and normal-appearing gray matter (NAGM) in MS patients. Postmortem brain tissue samples from 9 MS cases were compared with 10 controls using real-time polymerase chain reaction, immunoblot, and immunohistochemical analyses. Connexin32 and Cx47 gap junction formation in oligodendrocytes was reduced within lesions, whereas Cx32 loss also extended to NAGM. In contrast, astrocytic Cx30 expression was increased within cortical lesions, whereas Cx43 was elevated in both lesions and NAGM. Diffuse microglial activation and marked astrogliotic changes accompanied these connexin abnormalities. Increased expression of Cx43 correlated with inflammatory load (r = 0.828, p = 0.042), whereas Cx32 expression correlated with longer disease duration and, therefore, milder course (r = 0.825, p = 0.043). Thus, there is a loss of intramyelin and intercellular oligodendrocyte gap junctions in MS gray matter lesions and NAGM, whereas interastrocytic gap junctions are increased, reflecting astrogliosis. These changes correlate with inflammation and disease duration and suggest that disconnection of oligodendrocytes from reactive astrocytes may play a role in failed remyelination and disease progression.