We studied the pathway of 5S ribosomal RNA (rRNA) during oogenesis in Xenopus from its storage in the cytoplasm to incorporation into ribosomes in the nucleus. Ribonucleoprotein particle (RNP) assembly assays reveal striking differences in the behavior of oocyte-type and somatic-type 5S rRNA after microinjection into stage II, III, or IV oocytes or into the cytoplasm of stage V-VI oocytes. Microinjected oocyte-type 5S rRNA predominantly interacts with the 5S rRNA gene-specific transcription factor IIIA (TFIIIA) to form storage 7S RNPs. In contrast, microinjected somatic-type 5S rRNA predominantly interacts with ribosomal protein L5 to form 5S RNPs, which are precursors to ribosome assembly. In addition, a greater amount of somatic-type 5S rRNA accumulates in the nucleus and is assembled into 60S ribosomal subunits. Thus, a slight difference in nucleotide sequence results in differential binding of 5S rRNA to TFIIIA and L5, specializing oocyte-type for storage in the oocyte cytoplasm and somatic-type for rapid mobilization and ribosome assembly. When oocyte-type and somatic-type 5S rRNA molecules were microinjected into the nucleus of stage V-VI oocytes in excess of other ribosomal components, the nucleocytoplasmic distribution of both types of RNA was similar, but the distinctive protein associations were maintained. In contrast, the behavior of oocyte-type and somatic-type 5S rRNA gradually synthesized in situ from microinjected cloned genes was similar, suggesting that nascent RNA is rapidly and directly recruited into ribosomes, thus bypassing an excursion into the cytoplasm prior to ribosome assembly.