The locations of RNA-RNA cross-links in 16S rRNA were determined after in vivo incorporation of 4-thiouridine (s(4)U) into RNA in a strain of Escherichia coli deficient in pyrimidine synthesis and irradiation at >320 nm. This was done as an effort to find RNA cross-links different from UVB-induced cross-links that would be valuable for monitoring the 30S subunit in functional complexes. Cross-linked 16S rRNA was separated on the basis of loop size, and cross-linking sites were identified by reverse transcription, RNase H cleavage, and RNA sequencing. A limited number of RNA-RNA cross-links in nine regions were observed. In five regions-s(4)U562 x C879-U884, s(4)U793 x A1519, s(4)U1189 x U1060-G1064, s(4)U1183 x A1092, and s(4)U991 x C1210-U1212-the s(4)U-induced cross-links are similar to UVB-induced cross-links observed previously. In four other regions-s(4)U960 x A1225, s(4)U820 x G570, s(4)U367 x A55-U56, and s(4)U239 x A120-the s(4)U-induced cross-links are different from UVB-induced cross-links. The pattern of cross-linking is not limited by the distribution of s(4)U, because there are at least 112 s(4)U substitution sites in the 16S rRNA. The relatively small number of s(4)U-mediated cross-links is probably determined by the organization of the RNA in the 30S subunit, which allows RNA conformational flexibility needed for cross-link formation in just a limited region.