The small ribonucleoproteins recognized by anti-La autoantibodies contain a heterogeneous mixture of small RNAs from uninfected mammalian cells. The identity of many of these has now been established by the discovery of precursor forms of 5S rRNA and of certain tRNAs among La RNAs from HeLa cells. The small fraction of 5S rRNA molecules that exist as La ribonucleoproteins in vivo possess 1 or 2 additional U residues at their 3' ends. Such 5S molecules bound to the La protein have also been identified with in vitro nuclear transcription systems. Pulse-chase experiments performed both in vivo and in vitro support the idea that most newly synthesized 5S rRNA molecules are transiently associated with the La protein. Cell extracts contain a processing activity that converts longer in vitro-synthesized 5S RNA transcripts into molecules of mature size. The presence of in vivo tRNA precursors in the heterogeneous mixture of La RNAs is demonstrated by the identification of precursor forms of five different specific tRNAs (Meti, Asp, Gly, Glu, Asn). After in vitro transcription of a tRNA gene (tRNAiMet), only products the size of precursor molecules are precipitable by anti-La antibodies. The realization that virtually every known RNA polymerase III product associates at least initially with the La antigen suggests that this protein plays an essential role in the synthesis or maturation of all class III transcripts.