The heat-induced melting of yeast 5S RNA and tRNAPhe has been monitored by UV, CD, and 360-MHz 1H NMR spectroscopy in order to determine the extent of base stacking and base pairing in the native and denatured structures. In the presence of Mg2+, the optical data indicate less than or equal to 40 base pairs in native yeast 5S RNA, a 60:40 ratio of GC to AU base pairs, with more single-stranded stacking and a slightly less stable structure (half-melted at 67 degrees C) than for tRNAPhe (half-melted at 71 degrees C). In the absence of Mg2+, the NMR results identify a minimum of approximately 32 base pairs at 25 degrees C (increasing to a minimum of approximately 35 base pairs in the presence of Mg2+), of which more than half are still intact at 48 degrees C. The native structure (25 degrees C) shows only minor dependence upon Mg2+ concentration, and no denatured forms could be detected. Finally, the present results support a previously proposed cloverleaf secondary structure for eukaryotic 5S RNA.