Inelastic electron tunneling spectroscopy (IETS) was performed on single molecules with a variable temperature scanning tunneling microscope. The peak intensity, width, position, and line shape of single molecule vibrational spectra were studied as a function of temperature, modulation bias, bias polarity, and tip position for the (C–H,C–D) stretching vibration of acetylene (C2H2,C2D2) on Cu(001). The temperature broadening of vibrational peaks was found to be a consequence of Fermi smearing as in macroscopic IETS. The modulation broadening of vibrational peaks assumed the expected form for IETS. Extrapolation of the peak width to zero temperature and modulation suggested an intrinsic width of ∼ 4 meV due primarily to instrumental broadening. The inelastic tunneling cross section at negative bias was reduced by a factor of 1.7 for the C–H stretch mode. Low energy modes of other molecules did not show such a reduction. There was no evidence of a tip-induced Stark shift in the peak positions. The spatial variation of the inelastic signal was measured to determine the junction stability necessary for the acquisition of single molecule vibrational spectra. © 2001 American Institute of Physics.