Anti-La antibodies are frequently found in patients with autoimmune diseases; the antigen was reported to be a 50,000-Da protein (Rinke, J., and Steitz, J. A. (1982) Cell 29, 149-159). Because this protein was associated with many nascent RNA polymerase III transcripts, it was suggested to be an RNA polymerase III transcription factor. The present study was designed to analyze 4.5 I ribonucleoprotein, an RNA polymerase III transcript which contains the La antigen. It was found that the 3'-end 20-30-nucleotide portion was the most protected portion of 4.5 I RNA when 4.5 I ribonucleoprotein was digested with T1 RNase. When U2 RNA (an RNA polymerase II transcript) and 4.5 I RNA were incubated with the S-100 fraction of Novikoff hepatoma cells, the 4.5 I RNA bound La antigen but the U2 RNA did not. When partial and complete T1 RNase digestion fragments of 4.5 I RNA were incubated with the S-100 fraction, the 3'-end fragments bound preferentially to the La antigen. However, the fragments of 4.5 I RNA bound less efficiently to La antigen than whole 4.5 I RNA. These results indicate that the 3'-end of 4.5 I RNA is the La antigen binding site in this molecule and suggest that the overall conformation of RNA aids in the binding of La antigen.