The 16S rRNA central pseudoknot region in the 30S ribosomal subunit has been investigated by photocrosslinking from 4-thiouridine (s4U) located in the first 20 nt of the 16S rRNA. RNA fragments (nt 1–20) were made by in vitro transcription to incorporate s4U at every uridine position or were made by chemical synthesis to incorporate s4U into one of the uridine positions at +5, +14, +17, or +20. These were ligated to RNA containing nt 21–1542 of the 16S rRNA sequence and, after gel purification, the ligated RNA was reconstituted into 30S subunits. Long-range intramolecular crosslinks were produced by near-UV irradiation; these were separated by gel electrophoresis and analyzed by reverse transcription reactions. A number of crosslinks are made in each of the constructs, which must reflect the structural flexibility or conformational heterogeneity in this part of the 30S subunit. All of the constructs show crosslinking to the 559–562, 570–571, and 1080–1082 regions; however, other sites are crosslinked specifically from each s4U position. The most distinctive crosslinking sites are: 341–343 and 911–917 for s4U(+5); 903–904 (very strong), 1390–1397, and 1492 for s4U(+14); and 903–904 (moderate) for s4U(+17); in the 1070–1170 region in which there are different patterns for each s4U position. These results indicate that part of the central pseudoknot is in close contact with the decoding region, with helix 27 in the 885–912 interval and with part of domain III RNA. Crosslinking between s4U(+14) and 1395–1397 is consistent with base pairing at U14-A1398.