The 23S rRNA gene of Chlamydomonas reinhardtii contains a group IA3 intron, Cr.LSU, whose splicing is essential for cell growth. To better understand Cr.LSU splicing, kinetic analyses were undertaken with 23S.3, a preRNA previously shown to self-splice. Self-splicing of 23S.3 showed biphasic kinetics, with only similar to 33% reacting efficiently. Removal of a region of the 5' exon that could potentially interfere with the intron core (i.e., P3) increased the size (53%) of the active fraction. Replacement of the large P6a-extension by a 20-nt stem-loop further increased the active fraction to similar to 80%. The k(cat) and K-M(G) for self-splicing (first step) by these latter RNAs were similar to 1 min(-1) and similar to 20 mu M, respectively, These results indicate that Cr.LSU is a highly efficient ribozyme whose folding in vitro is impeded by exonic and/or intronic sequences. The implications for in vivo splicing of Cr.LSU are discussed.