We report a comparative study of the structure and chemistry of methyl-terminated n-alkanethiol self-assembling monolayers (SAMs) formed from liquid and vapor phases. Three different SAMs are considered : Au/HS(CH2)nCH3, n = 5, 11, and 15. Liquid-phase-deposited films were prepared by exposure of Au substrates to dilute ethanol solutions of the n-alkanethiols followed by ethanol rinsing, and vapor-phase-deposited SAMs were prepared by exposure of the Au surface to 10%-of-saturation n-alkanethiol vapors followed by N2 purging, which removes loosely bound n-alkanethiol molecules from the surface. The matrix of six organic surfaces was studied by FTIR external reflectance spectroscopy (FTIR-ERS), ellipsometry, and cyclic voltammetry, which provide information about the average structure of the SAMs, and a newly developed scanning tunneling microscope (STM)-based method, which provides information about individual SAM defect structures. FTIR-ERS and ellipsometry indicate no detectable differences between liquid- and vapor-phase-deposited SAMs. Data obtained using cyclic voltammetry and STM show that the barrier properties of SAMs depend on the ambient phase from which the SAM assembles, the length of the n-alkanethiol, and the chemical nature of the molecular probe used to evaluate the monolayer structure. For example, vapor-phase-deposited Au/HS(CH2),11CH3 SAMs are better CN- mass-transfer barriers than their liquid-phase-deposited analogs. However, Au/HS(CH2)11CH3 SAMs are better CN-barriers when they are formed from the liquid phase. In contrast, Au/HS(CH2)11CH3 SAMs prepared from either phase present nearly identical barriers to electron exchange between the Au surface and solution-phase Ru(NH3)63+. STM reveals some of the nanostructural details of SAMs and confirms that individual defects govern their barrier properties.