OBJECTIVE Several studies have indicated the anatomic and biochemical presence of gap junctions in corneal keratocytes. The current study was designed to demonstrate that these gap junctions are functional in rabbit and human corneal keratocytes. This study also examined dye coupling between keratocytes migrating into the wound region of freeze-wounded rabbit corneas. METHODS Freeze wounds were created on anesthetized rabbit corneas using a liquid nitrogen-cooled brass probe. Freeze-wounded corneas were examined at several time periods from days 0 to 5 after wounding. Nonwounded rabbit corneas also were examined. Human corneal buttons were examined immediately after removal from patients who underwent keratoplasty. Gap junctional coupling was examined by microinjecting carboxyfluorescein from microelectrodes into the basal-most keratocytes and capturing dye spread images with a cooled charge coupled device camera. RESULTS Significant dye spread was observed between cells in the unwounded areas of corneas at wound time 0 and between cells migrating into the wound areas as early as 24 hours after wounding. In control corneas, dye spread to as many as 50 cells from the source cell. Dye spread also was seen between keratocytes in human corneas with pseudophakic bullous keratopathy and keratoconus. CONCLUSIONS Gap junctions observed in keratocytes from normal rabbit corneas are functional. Gap junctions also are present and functional in keratocytes within unwounded and wounded regions of freeze-injured corneas. In addition, functional gap junctions are present between keratocytes in human corneas. This study confirms the long-held contention that corneal keratocytes form a large intercommunicating network within the corneal stroma.