Group I introns constitute excellent systems for analyzing the relationship between RNA tertiary folding and catalysis. Within a hierarchical framework interpretation of RNA folding, secondary structure motifs subtend RNA three-dimensional (3D) architecture. Thus, mutations in two-dimensional motifs are expected to have effects different from those disrupting 3D contacts. Using UV spectroscopy, we have studied the influence of nucleotide substitutions, in both secondary and tertiary structure elements, on the thermal stability of the tertiary folding of the bacteriophage T4 td group I intron. Further, we present a quantitative analysis of the relationship between the splicing efficiency in vivo and the stability of the intron structure as monitored by UV melting curves. We conclude that the stability of the tertiary structure of a group I intron as measured by UV melting is generally a good indication of its ability to splice in vivo.