L-21 ScaI ribozyme is a linear form of the self-splicing intron from the precursor of the Tetrahymena thermophila LSU intron. The time scales for tertiary folding of L-21 ScaI were investigated in two ways after bringing it from a partially denatured state at 60 degrees C and 1 mM Mg2+ to a renatured state of 15 degrees C and 10 mM Mg2+. First, formation of a catalytically active structure was monitored by measuring the kinetics of the reaction: p*CUCUA3 + G<==>p*CUCU + GA3. This reaction mimics the first step of splicing. After 1 min of folding time, the catalytic rate is roughly 10% of the rate attained after 8 h of folding. This indicates that much of the structure refolds quickly. Also, at least two time scales of folding are observed, separated by a lag time of about 30 min. To define the regions folding on various time scales, all the guanosines of L-21 ScaI were probed with kethoxal at 15 degrees C while folding was in progress. Based on folding time scales, the guanosines can be placed into at least four classes. These are guanosines that (1) are already protected at 60 degrees C in 1 mM Mg2+ or which fold immediately, (2) fold during the lag time, (3) continue to fold after 1 h, and (4) never fold. These results give insight into the folding pathway of a group I ribozyme at nucleotide resolution. This provides useful information on the regions whose foldings are important for catalytic function of the ribozyme. The method may also provide a general way to suggest regions of an RNA that may interact with each other to form tertiary structure.