We have found that a solvent mixture of ethanol and dodecane was able to disperse natural ceramide (Cer) into aqueous solution. In U937 cells, addition of natural Cer to medium caused a characteristic pattern of DNA fragmentation, which is indistinguishable from that caused by TNF alpha. The effective concentration of Cer is as low as 25 nM. The present study provides direct evidence that natural Cer functions as a second messenger mediating TNF alpha-induced DNA fragmentation. The use of this solvent to deliver hydrophobic natural Cer to cells will contribute to the elucidation of the biological function of Cer.