Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences: (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis.