A peptide corresponding to the N-terminal sequence of the rat malate dehydrogenase, comprising the transit sequence and two residues of the mature protein (MLSALARPVGAALR-RSFSTSAQNNAK) has been chemically synthesized, and its structural characteristics investigated by Fourier-transform i.r. (FT-IR), c.d. and 1H-n.m.r. spectroscopy. FT-IR and c.d. spectra of the peptide were recorded in a variety of environments (aqueous solution, trifluoroethanol) and after incorporation into phospholipid bilayers. The peptide was found to be mainly in aperiodic or undefined conformation in aqueous solution. However, in trifluoroethanol a marked increase in alpha-helical content was observed. An increase in alpha-helical content was also observed in negatively charged lipids (dimyristoylphosphatidylglycerol and cardiolipin). However, when reconstituted in a zwitterionic phospholipid (dimyristoylphosphatidylcholine), no alpha-helical structure was observed. N.m.r. spectroscopy was used to characterize the helical structure in greater detail in trifluoroethanol. The 1H-n.m.r. spectrum of the peptide in this solvent was assigned using standard homonuclear two-dimensional methods. The observed patterns of nuclear Overhauser enhancements confirmed the deductions obtained from c.d. and FT-1R spectroscopy concerning the solution conformation, suggesting a region of flexible nascent helix between Ala-4 and Ser-18. This structure is discussed in terms of the possible function of the peptide.