An immediate precursor of 5S ribosomal RNA (rRNA) from Bacillus subtilis has 21 and 42 nucleotide precursor-specific segments associated with its 5' and 3' termini, respectively. On the basis of its nucleotide sequence, predicted secondary structure and location in the rRNA transcriptional unit, the 3' precursor element apparently functions during the termination of transcription. A portion of the 5' precursor element is shown to facilitate the native folding of the mature domain of the precursor. Precursor 5S rRNA molecules which lack the 5' terminal 8-9 nucleotides of the 5' precursor elements were fabricated. These abbreviated constructs assume a non-native conformation, as revealed by their behavior during polyacrylamide gel electrophoresis. The aberrant conformation is evidently forced upon the abbreviated constructs by the residual 5' precursor sequence, since its removal by the maturation endonuclease RNAase M5 precipitates the reordering of the mature domain into its native conformation. Inspection of the nucleotide sequence of the 5S precursor suggested the nature of the conformational aberration, and gel electrophoresis analyses of limited nuclease digests of end-labeled precursors in the native and aberrant conformations are consistent with the derived model. We conclude taht the 5' terminal six nucleotides in the intact 5S precursor assist in the folding of the mature domain by forming a base-paired duplex with neighboring nucleotides, thereby preventing that adjacent sequence from engendering the abnormal conformation. The involvement of precursor-specific sequences and conformational dynamics in RNA function are discussed.