Non-ionic surfactant vesicles (niosomes) have shown promise as cheap, chemically stable alternatives to liposomes. Niosomes of spans (Sorbitan monoesters) have shown promise of commercial exploitation. Hence, niosomes were prepared of 5-fluorouracil (FU) using different spans. Niomsomes were prepared by the hand shaking method (HSM), reverse phase evaporation (REV) and ether injection method (EIM) using a series of Spans, i.e. Span 20, 40, 60 and 80. HSM giving least permeable vesicles were used to study the effect of variables like type of Span, composition of lipid and total lipid concentration on entrapment efficiency (EE) and release rate. Span 40 and 60 released 40.9 and 37.1% drug in 6 h while Span 20 and 80 displayed 52.2 and 57.1% release, respectively in the same time. Niosomes of Span 40 showed a mean vesicle size of 8.1 mu m, EE of 15.3 +/- 1.3% and released 78.6% drug in 6 h; inclusion of cholesterol reduced the vesicle size to 4.8 mu m, EE to 12.3 +/- 0.9% and the release to 50.5% tin 6 h), while incorporation of dicetyphosphate further reduced the vesicle size to 3.87 mu m, EE to 10.9 +/- 1.1% and reduced release to 40.9% (in 6 h). Increase in the amount of lipid used translated into an almost linear increase in EE. Biodistribution of drug in rats was modified on encapsulation. The concentration of niosomal drug in liver, lung and kidney was increased while it decreased in intestine compared to free drug solution following intravenous administration. The niosomal formulation displayed higher and sustained plasma drug level profile compared to free drug solution. Pharmacokinetic calculations revealed an increase in half-life, area under the curve and decrease in volume of distribution of the drug on encapsulation. Thus, the study suggests that niosomes can act as promising carriers for 5-Fluorouracil.