Different conformational states of Escherichia coli 5S ribosomal RNA that may participate in protein biosynthesis have been either detected experimentally or predicted on the basis of phylogenetic sequence comparisons. The A conformer exists in a high-salt form (AH) that binds ribosomal proteins and assembles into the 50S subunit and in a low-salt form (AL), of uncertain biological relevance, that binds at least one ribosomal protein and differs in tertiary structure from the AH form. Experimentally, the AH form has been investigated comprehensively and the AL form partially. There is also a B conformer that exhibits an altered secondary structure and does not assemble with ribosomal proteins. For this conformer exhibits an altered secondary structure and does not assemble with ribosomal proteins. For this conformer to be functionally active, it must be both discrete and universal among 5S RNAs. Here, we examine its structure by employing single and double strand specific ribonucleases and nucleotide-specific chemical reagents. We demonstrate that the B form exhibits a secondary structure only a part of which is both universal and conformationally homogeneous, and we conclude, therefore, that the whole B form cannot participate in protein biosynthesis. We note, however, that progressive structural changes occur during the transitions AH----AL----B and provide evidence that the structural alteration during the transition AH----AL may be universal, which reinforces the view that the AL form is of biological relevance.