It is assumed that the primary mode of action of 5-fluorouracil (5-FUra) is mediated via inhibition of thymidylate synthetase. Persistent inhibition of cellular proliferation after treatment of the 5-FUra-inhibited cells with exogenous thymidine do not support the notion that the anti-proliferitive action of 5-FUra is due exclusively to inhibition of DNA replication. Our studies have revealed an alternative mechanism of action at the level of pre-ribosomal RNA (pre-rRNA) processing. Pre-rRNA processing was inhibited completely in vitro as well as in S-100 extract from the mouse lymphosarcoma P1798 cells that were treated with 5-FUra. Under this condition, the 5-FUra-substituted pre-rRNA substrate was processed efficiently at the primary processing site. This study showed that the activity and/or the synthesis of a factor potentially involved in pre-rRNA processing is blocked in cells treated with the fluoropyrimidine. UV-cross-linking study showed that a 200 kDa polypeptide designated ribosomal RNA binding protein (RRBP) was absent in the S-100 extract from the drug-treated mouse lymphosarcoma cells. Since a polypeptide that cross-links to a processing site on RNA is usually involved in the RNA processing, RRBP may have a direct role in pre-rRNA processing. A key molecular mechanism far the antiproliferative action of 5-FUra may be due to its interference with the activity and/or synthesis of RRBP. Exposure of cells to 5-FUra did not inhibit the interaction between U3 small nucleolar RNA (snoRNA) and pre-rRNA at the primary processing site (a key step in the processing reaction) and the formation of U3 small nucleolar ribonucleoprotein (snoRNP). Treatment of cells with the fluoropyrimidine did not block the 3' end processing of pre-messenger RNA (pre-mRNA). This article also discusses the effects of 5-FUra on pre-mRNA splicing and mRNA translation, and proposes other avenues of research to explore further the mechanism of action of this important pyrimidine analog.