The cathepsin L gene of the host squid， Euprymna scolopes， is upregulated during the first hours of colonization by the symbiont Vibrio fischeri. At this time， the symbiotic organ begins cell death-mediated morphogenesis in tissues functional only at the onset of symbiosis. The goal of this study was to determine whether Cathepsin L， a cysteine protease associated with apoptosis in other animals， plays a critical role in symbiont-induced cell death in the host squid. Sequence analysis and biochemical characterization demonstrated that the protein has key residues and domains essential for Cathepsin L function and that it is active within the pH range typical of these proteases. With in situ hybridization and immunocytochemistry， we localized the transcript and protein， respectively， to cells interacting with V. fischeri. Activity of the protein occurred along the path of symbiont colonization. A specific Cathepsin L， nonspecific cysteine protease， and caspase inhibitor each independently attenuated activity and cell death to varying degrees. In addition， a specific antibody decreased cell death by ~50%. Together these data provide evidence that Cathepsin L is a critical component in the symbiont-induced cell death that transforms the host tissues from a colonization morphology to one that promotes the mature association.