The present study was designed to investigate the role of skin sphingosine inhibition in enhancing the transcutaneous permeation of 5-fluorouracil (5-FU), a hydrophilic drug, across rat skin. Ethanol-perturbation significantly reduced the sphingosine content as compared to that in normal skin until 24 h of perturbation (p < 0.05). To maintain the low content of sphingosine for a longer time, beta-chloroalanine (beta-CA), a selective inhibitor of serine palmitoyl transferase, was used for inhibiting the sphingosine synthesis in viable skin. Application of beta-CA (600 microg or 1200 microg/7 cm2) to viable skin perturbed with ethanol significantly reduced the sphingosine content until 48 h as compared to that in perturbed viable skin (p < 0.05). However, the sphingosine content in viable skin perturbed with ethanol and treated with lower doses (200 or 400 microg/7 cm2) of beta-CA, returned significantly close to that in ethanol-perturbed viable skin at 36 h (p < 0.05). Skin sphingosine synthesis inhibition efficacy of 1200 microg beta-CA was insignificantly different to that of 600 microg dose of beta-CA at 36 h or 48 h (p < 0.05). The systemic delivery of percutaneously applied 5-FU across ethanol-perturbed rat skin treated with either 600 microg or 1200 microg beta-CA was significantly greater as compared to that obtained after oral administration or after application of lower percutaneous doses of beta-CA (p < 0.05). Higher Cmax, MRT, AUC and maintenance of effective plasma concentration of 5-FU for 46 h was achieved by a single topical application of a formulation containing 5-FU and 600 microg beta-CA to ethanol-perturbed skin.