16S rRNA reacted with the furocoumarin 4,5',8-trimethylpsoralen (trioxsalen) and 360-nm light showed a number of chemical and physical differences from untreated RNA. After extensive irradiation, five molecules of trioxsalen were bound per molecule of RNA. The trioxsalen-treated RNA had an altered ultraviolet absorption spectrum and a distinctive fluorescence emission spectrum. The modified RNA was significantly more resistant to T1 ribonuclease digestion than was control RNA. Treated RNA, when mixed with purified ribosomal proteins, was not functional in the in vitro reconstitution of 30S subunits and yielded more slowly sedimenting particles which were inactive in protein synthesis assays. By contrast, 16S rRNA within the 30S subunit structure did not exhibit these changes when reacted with the same dose of trioxsalen and light, suggesting that the ribosomal proteins were effective in protecting the RNA from interaction with the drug.