Combined DEAE-Sephacel and hydroxyapatite chromatography resulted in a separation of free Sm antigen from the Sm/RNP complex in rabbit thymus extracts. In Western blots, the free Sm preparation contained an immunoreactive 14-kd (D) protein, whereas the S d R N P complex contained, in addition to the 14-kd protein, a 68-kd-reactive protein and its putative degradation fragments. Ethidium bromide staining of these preparations separated by agarose gel electrophoresis showed that the S d R N P preparation contained RNA, but the free Sm preparation did not. These preparations were used as antigens in an enzyme-linked immunosorbent assay (ELISA). Sera characterized as anti8m only, anti-SdRNP, and anti-RNP only were assayed. A quotient (Q) was derived from the ELISA optical density obtained with Sm/RNP as antigen divided by the optical density obtained with free Sm as antigen. Q values <4.0 characterized sera with anti-Sm only, values of 4.0-12.0 were observed in sera with anti-Sm plus anti-RNP, and Publication number 4406BCR from the