Using immunological methods, we have identified parvalbumin-like material in the triclade flatworms, Polycelis nigra, Polycelis auriculata, Crenobia alpina, Dugesia tahitiensis, and Dugesia polychroa. 2. Western immunoblot analysis of these five species revealed heat stable parvalbumin-immunoreactive proteins between 32 kD and 44 kD. 3. Proteins at 19 and 32 kD revealed intense labeling with 45Ca2+. 4. Double immunodiffusion of planarian supernatants showed complete fusion of precipitates, indicating immunological relatedness of the parvalbumin-like material among the species investigated. 5. Immunocytochemical studies exhibit parvalbumin-immunoreactive material exclusively in neurons supporting the notion of an early evolutionary appearance of these proteins in the nervous system.