Binding of DNA to dimyristoylphosphatidylcholine (DMPC) liposomes containing different sphingosine derivatives was investigated. DNA labelled with adriamycin was used as a fluorescence quencher and its membrane association was observed by resonance energy transfer from liposomes incorporating a pyrene-derivatized lipid bisPDPC as a donor and containing 19 mol% of sphingosine, dihydro-, phyto- or dimethylsphingosine. As revealed by differential scanning calorimetry, the thermal phase behaviour of multilamellar liposomes containing these sphingolipids was also significantly altered by DNA. Attachment of DNA to liposomes containing sphingosylphosphorylcholine was much weaker, and no binding of DNA to membranes containing N-acetylsphingosine, N-stearoylsphingosine or sphingomyelin was observed. The membrane binding of DNA was dependent on pH and could be reversed by the inclusion of phosphatidic acid (eggPA) into the liposomes. Analogously, the association of cytochrome c with eggPA could be reversed by the DNA-binding sphingosines. These findings lend support to our previous proposal that the DNA-sphingosine interaction is electrostatic and requires the presence of a positive charge in the latter. Accordingly, sphingosines carrying a protonated amino group attach DNA to membranes, while blocking of the amino group by N-acylation abolishes this interaction.