A clear viscous fluid phase has been aspirated by renal micropuncture techniques from the predentinal space in 5-day-old rats from molars. Origin of the fluid will be studied by injection of a dye, 8-amino acridine, into the site of aspiration. Next, serial sections after fixation, dehydration, embedding and sectioning will be studied under fluorescent microscopy as already carried out for localization of Cf1 origin in cartilage micropuncture sites. Characterization of this fluid in relationship to electrolytes, Na, K, Cl, HCO3, Mg, Ca and Pi is underway. Free calcium and free phosphate will also be studied by an equilibrium dialysis method. We will attempt to characterize proteoglycans, glycoproteins and phosphoproteins in the fluid to the extent that the samples permit. It is proposed to study the proteins in the fluid by ultramicro two-dimensional disc gel electrophoresis with and without SDS inclusion. Large pore gels can be used for larger proteins and S value profiles obtained by analytical ultracentrifugation for proteoglycans. Various phosphohydrolases will be studied in relationship to wheather phosphoprotein, nucleotide, or evidence of phospholipids are obtained in the fluid. In an in vitro mineralizing system, evidence for nucleational properties for new mineral phase and analysis for inhibitors will be made. Methods used for studying cartilage micropuncture fluid will be used in these assessments for nucleators and inhibitors in predentinal fluid.