Voltammetric and in situ STM studies were carried out for self-assembled monolayers of 4-mercaptopyridine 4-PySH), 2-mercaptopyridine (2-PySH) and thiophenol (PhSH) on well-defined single-crystal Au(111) electrodes in aqueous solutions. A reversible voltammetric response for cytochrome c was clearly observed only at the 4-PyS/Au(lll) electrode, showing that only the 4-pyridinethiolate monolayer promotes facial electron transfer reaction between the Au(lll) and cytochrome c. On the basis of reductive desorption, the surface coverages of the three aromatic thiolate monolayers were found to be similar to each other; 4.6 x 10(-10) mol/cm(2) for 4-PyS/Au(lll), 4.7 x 10(-10) mol/cm(2) for 2-PyS/Au(lll), and 4.4 x 10(-10) mol/cm(2) for PhS/Au(lll). High-resolution STM images in perchloric acid solutions revealed p(5 x root 3R-30 degrees) and p(4 x root 7R-40.9 degrees) structures for the 4- and 2-pyridinethiolate monolayers on Au(lll), respectively. No structure order was observed for the PhSH monolayers. While the pyridine units of both 4- and 2-pyridinethiolate monolayers were found to be oriented normal to the surface, 2-pyridinethiolates adsorbed through not only sulfur but also nitrogen atom of the pyridine ring. From these STM images, the orientation of the N atom of the pyridine moiety must face to the bulk solution, as in the case of 4-PyS/Au(lll), in order to obtain a facile electrochemical reaction for cytochrome c.