The chain length dependence of helix formation of transmembrane peptides in lipids was investigated using fragments corresponding to the second transmembrane domain of the -factor receptor from Saccharomyces cerevisiae. Seven peptides with chain lengths of 10 (M2-10; FKYLLSNYSS), 14 (M2-14), 18 (M2-18), 22 (M2-22), 26 (M2-26), 30 (M2-30) and 35 (M2-35; RSRKTPIFIINQVSLFLIILHSALYFKYLLSNYSS) residues, respectively, were synthesized using solid phase chemistry and purified to near homogeneity. Circular dichroism (CD) spectra revealed that M2-10 was disordered and all the other peptides assumed partially -helical secondary structures in 99% trifluoroethanol (TFE)/H2O. In 50% TFE/H2O, M2-30 assumed a -like structure. The other six peptides exhibited the same CD patterns as those found in 99% TFE/H2O. In 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG) [4:1 ratio] vesicles M2-22, M2-26, and M2-35 formed -helical structures, whereas the other peptides formed -like structures. Fourier transform infrared spectroscopy and amide proton H/D exchange experiments showed that 64-95% of the NH bonds in these peptides did not exchange with bulk water in DMPC/DMPG (4:1) multilayers and that M2-10, M2-14, M2-18 and M2-30 assumed -structures in this environment. Another homologous 30 residue peptide (M2-30B), missing residues SNYSS from the N-terminus and extending to RSRKT on the C-terminus, was helical in lipid bilayers suggesting that residues at the termini of transmembrane domains influence their biophysical properties. Attenuated total reflection Fourier transform infrared spectroscopy revealed that M2-22, M2-26, M2-30B and M2-35 were -helical and oriented at angles of 12°, 13°, 36° and 34°, respectively, with respect to the multilayer normal. This study showed that chain length must be taken into consideration when using peptides representing single transmembrane domains as surrogates for regions of an intact receptor. Furthermore, this work indicates that the tilt angle and conformation of transmembrane portions of G protein-coupled receptors may be estimated by detailed spectroscopic measurements of single transmembrane peptides.