RNA processing depends in part on the ability of nascent transcripts to fold into the desired conformation. Self-splicing of the group I intron from Tetrahymena was used to assess the folded state of preribosomal RNA transcripts when synthesized in vitro. A simple method for isolating nondenatured RNA from a T7 RNA polymerase reaction was tested. The intron alone is fully active when transcribed at 30 degrees C, suggesting that the active structure is both kinetically and thermodynamically favored. Longer precursor RNAs, however, were less than completely active in self-splicing. Full activity, as judged by both the initial rate and the extent of product formation, was restored by brief incubation at 95 degrees C and rapid cooling in the presence of magnesium ion. This result did not depend on the length of the precursor RNA in any simple way, but correlated loosely with the presence of intact exon domains. When transcribed in the absence of cellular proteins, a significant portion of the pre-RNA appears to be trapped in a conformation that does not readily undergo the first step of splicing.