The mechanisms underlying resealing of transected myelinated rat dorsal root axons were investigated in vivo using an assay based on exclusion of a hydrophilic dye (Lucifer Yellow-biocytin conjugate). Smaller caliber axons (<5μm outer diameter) resealed faster than larger axons. Resealing was Ca 2 + dependent, requiring micromolar levels of extracellular [Ca 2 + ] to proceed, and further accelerated in 1mM Ca 2 + . Two hours after transection, 84% of axons had resealed in saline containing 2mM Ca 2 + , 28% had resealed in saline containing no added Ca 2 + and only 3% had resealed in the Ca 2 + buffer BAPTA (3mM). The enhancing effect of Ca 2 + could be overcome by both non-specific cysteine protease inhibitors (e.g., leupeptin) and inhibitors specific for the calpain family of Ca 2 + -activated proteases. Resealing in 2mM Ca 2 + was not inhibited by an inhibitor of phospholipase A 2 . Resealing in low [Ca 2 + ] was not enhanced by agents which disrupt microtubules, but was enhanced by dimethylsulfoxide (0.5-5%).These results suggest that activation of endogenous calpain-like proteases by elevated intra-axonal [Ca 2 + ] contributes importantly to membrane resealing in transected myelinated mammalian axons in vivo.