Photocrosslinking has identified the joiner between domains 2 and 3 [J(23)] as folding near domain 5 (D5), a highly conserved helical substructure of group II introns required for both splicing reactions. D5 RNAs labeled with the photocrosslinker 4-thiouridine (4sU) reacted with highly conserved nucleotides G588 and A589 in J(23) of various intron acceptor transcripts. These conjugates retained some ribozyme function with the lower helix of D5 crosslinked to J(23), so they represent active complexes. One partner of the gamma x gamma' tertiary interaction (A587 x U887) is also in J(23); even though gamma x gamma' is involved in step 2 of the splicing reaction, D5 has not previously been found to approach gamma x gamma'. Similar crosslinking patterns between D5 and J(23) were detected both before and after step 1 of the reaction, indicating that the lower helix of D5 is positioned similarly in both conformations of the active center. Our results suggest that the purine-rich J(23) strand is antiparallel to the D5 strand containing U32 and U33. Possibly, the interaction with J(23) helps position D5 correctly in the ribozyme active site; alternatively, J(23) itself might participate in the catalytic center.