We have analyzed RNP formation and nucleocytoplasmic migration of 5S RNA and 5S RNA variants transcribed from microinjected genes in Xenopus oocytes. Using antisera against three different proteins we find that newly transcribed nuclear 5S rRNA transiently interacts with La antigen. The La protein is then replaced by either ribosomal protein L5 or the 5S gene-specific transcription factor IIIA (TFIIIA), and each of these two RNPs migrates out of the nucleus and accumulates in the cytoplasm. RNA molecules that are impaired in their ability to interact with L5 and TFIIIA are retained in the nucleus. Thus, L5 and TFIIIA define a new functional class of proteins involved in the nuclear export of RNA. In addition, we show that RNP migration depletes the nucleus of TFIIIA, resulting in a loss of transcription competence for newly injected 5S rRNA genes.