Basic characteristics of calretinin (CR) and related neuronal calcium binding proteins identified under the project are used to learn about intracellular sites and modes of action. (1) Results revealed that the ability of antibodies to bind to calcium binding proteins depends on the calcium binding status, and thus, on the protein conformation at the time of fixation. A new antibody was developed to CR which recognizes CR in both calcium bound and unbound conformations to an equivalent extent in formalin fixed samples. Comparisons were made of the immunoreactivity of this antibody with that of a conformation sensitive CR antibody on brain tissue culture following application of drugs which increase intracellular calcium. The results suggest that characterized antibodies to calcium binding proteins may be used to examine the calcium status of neurons at the time of formalin fixation. (2) Previous subcellular fractionation studies showed calcium dependent association of CR and calbindin D28k (CB) with membrane fractions of rat cerebellum while parvalbumin is not associated with membrane fractions. A study was undertaken to examine whether the membrane association of CR might be partly due to hydrophobic interactions with lipids present in membranes. Results revealed a calcium-dependent association of CR with phospholipid vesicles containing phosphatidyl inositol and phosphatidyl serine. Other charged phospholipids (ceramide, cardiolipin) did not interact with CR and samples of parvalbumin did not associate with the phospholipids. These results suggest that specific phospholipids may be targets of interaction of CR and possibly CB. These interactions could affect the cellular distribution of calcium and calcium-mediated signals.