The recently licensed pneumococcal conjugate vaccine contains 7 different types (4, 6B, 9V, 14, 18C, 19F, and 23F) conjugated to CRM197 protein. The immunochemical assay by rate nephelometry is being used for evaluation of individual PS content in polyvalent pneumococcal vaccines. Nephelometry measures the intensity of scattered light as it is passed through the antigen-antibody complex formed by the precipitin reaction. For quantitation of PS content by nephelometry, the optimum concentrations for PSs and antibodies need to be determined. The objective of the present study was therefore to determine the optimum conditions for quantitative determination of individual PS in a polyvalent PS-protein conjugate vaccine. A multivalent conjugate vaccine was treated with trypsin to digest the carrier protein, thus releasing PSs into solution. For reference, monovalent conjugate vaccines, 10 ug/ml, were treated with trypsin and the released PSs diluted to different concentrations.(0.5 to 10 ug/ml) The antisera used were type specific hyperimmune rabbit sera against each pneumococcal type. Standard curves for types 4, 6B, 9V, 14, 18C, 19F, and 23F PSs were proportional to the increase of PS concentration in the range of 0.5 to 10 ug/ml. An excess or high PS concentration resulted in inhibition of the precipitin reaction and a reduction in the Ag-Ab complexes and thereby a decreased scattering rate. The optimum concentration for these 7 PS types, displaying linearity when used with their respective antisera, was found to be 1-5 ug/ml. Rate nephelometry is simple in operation, utilizes small quantities of antigen and antibody, and yields reproducible and accurate data for the quantitative analyses of individual PSs in polyvalent vaccines. However, it requires the reference standards for PS antigens and specific high titered antisera. Substances and conditions that may interfere with the nephelometric immunoassay system are being examined.