We examined the mechanism by which capsaicin produces its toxic effects on cultures of rat sensory neurons. Capsaicin caused a robust increase in [Ca²⁺]_i in a subpopulation of cultured rat dorsal root ganglion neurons. Similarly, a brief exposure to capsaicin resulted in delayed degeneration of a subpopulation of the cells. This subpopulation (about 35% of the cells present) was characterized by a capsaicin-induced uptake of Co²⁺, which could be detected cytochemically. Both capsaicin-induced Co²⁺ uptake and capsaicin-induced cell death were blocked by the capsaicin antagonist Ruthenium Red. Cell death was also prevented by removal of external calcium or by inhibiting calcium-activated proteases such as calpain. Evidence that calpain activity was increased was provided by examining the amount of degradation of the preferred calpain substrate α-spectrin. Capsaicin treatment produced a significant increase in the levels of the 150,000 molecular weight spectrin breakdown product. Furthermore, applying the protease inhibitors E64 or MDL 28,170 reduced capsaicin-mediated cell death. It is concluded that capsaicin kills a subpopulation of sensory neurons by activating a receptor-operated channel. The consequent Ca²⁺ ion influx causes large increases in [Ca²⁺]_i and subsequent activation of Ca²⁺-sensitive proteases. This model provides support for the role of [Ca²⁺]_i as the orchestrator of delayed neuronal degeneration.