Chaperonin 10 (Cpn10) is one of only a few mitochondrial matrix proteins synthesized without a cleavable targeting signal. Using a truncated form of Cpn10 and synthetic peptides in mitochondrial import assays, we show that the N-terminal region is both necessary and sufficient for organellar targeting in vitro. To elucidate the structural features of this topogenic signal, peptides representing residues 1-25 of rat Cpn10 were synthesized with and without the naturally occurring N-terminal acetylation. 1H NMR spectroscopy in 20% CF3CH2OH,H2O showed that both peptides assume a stable helix-turn-helix motif and are highly amphiphilic in nature. Chemical shift and coupling constant data revealed that the N-terminal helix is stabilized by N-acetylation, whereas NOE and exchange studies were used to derive a three dimensional structure for the acetylated peptide. These findings are discussed with respect to a recent model predicting that targeting sequences forming a continuous alpha-helix of more than 11 residues cannot adopt a conformation necessary for proteolysis by the matrix located signal peptidases (Hammen, P. K., Gorenstein, D. G., and Weiner, H. (1994) Biochemistry 33, 8610-8617).