The spacing of chemical functional groups on self-assembled monolayers (SAMs) plays an important role in controlling the density of biomolecules in biochips and biosensors. In this sense, a mixed SAM made of two different terminal groups is a useful organic surface since spacing can be easily controlled by changing a relative mole fraction in a mixture solution. In this study, an acetylene-OCH2O(EG)(3)(CH2)(11)S-S(CH2)(11)(EG)(3)OCH2O-propene (Eneyne) SAM and mixed SAMs made by a mixture of (S(CH2)(11)(EG)(3)O-CH2O-acetylene)(2) (Diyne) and (S(CH2)(11)(EG)(3)OCH2O-propene)(2) (Diene) were produced on gold substrates and measured by using ToF-SIMS. The secondary ion yield ratio of [Au center dot S(CH2)(11)(EG)(3)O-11(EG)(3)OCH2O-acetylene] to [Au center dot S(CH2)(11)(EG)(3)OCH2O-propene] was measured for each mixed SAM and plotted as a function of the mole fraction of Diyne to Diene in a SAM solution. The ion yield ratio of a mixed SAM produced from a solution with a mole fraction of 0.5 (i.e., 1:1 mixture) was 0.3, which corresponded well to the ion yield ratio measured from an Eneyne SAM. A time-dependent experiment of Eneyne SAM formation and immersion experiment of Eneyne SAM into Diyne solution or into Diene solution were performed. The relative ion yield ratio of 0.3 was due to a different secondary ion formation and not due to the difference in the amount of adsorbates on the surface, nor to the different binding strengths onto the gold surface. Our study shows that a mixed SAM with well-controlled spacing can be produced and quantified by using the ToF-SIMS technique. (C) 2008 Elsevier B. V. All rights reserved.