Neuronal regeneration after damage to an axon tract requires the rapid sealing of the injured plasma membrane and the subsequent formation of growth cones that can lead regenerating processes to their appropriate target. Membrane sealing and growth cone formation are Ca²⁺‐dependent processes, but the signaling pathways activated by Ca²⁺ to bring about these effects remain poorly understood. An in vitro injury model was employed in which neurites from identified snail neurons (Helisoma trivolvis) were transected with a glass microknife, and the formation of new growth cones from the distal portions of transected neurites was recorded at defined times after transection. This study presents three main results. First, phospholipase A₂ (PLA₂), a calcium‐activated enzyme, is necessary for membrane sealing in vitro. Second, PLA₂ activity is also required for the formation of a new growth cone after the membrane has sealed successfully. Thus, PLA₂ plays a dual role by affecting both growth cone formation and membrane sealing. Third, the injury‐induced activation of PLA₂ by Ca²⁺ controls growth cone formation through the production of leukotrienes, secondary metabolites of PLA₂ activity. Taken together, these results suggest that the injury‐induced Ca²⁺ influx acts via PLA₂ and leukotriene production to assure growth cone formation. These findings indicate that events that cause an inhibition of PLA₂ or lipoxygenases, enzymes that produce leukotrienes, could result in the inability of neurites to regenerate. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 555–565, 2003