We report a quantitative study of the kinetics of formation for a two-component tethered ssDNA monolayer film using in situ two-color surface plasmon resonance (SPR) spectroscopy. The attachment of the DNA to gold is facilitated by functionalization at the 5′ end with a thiol group connected by a hexamethylene linker (HS-C6-ssDNA). Detailed data analysis is performed by quantitative comparison of the DNA coverage versus time kinetic data obtained from SPRS with numerical solutions for the differential equations for simultaneous adsorption, desorption, and diffusion at the interface. The kinetics of adsorption of HS-C 6 -ssDNA onto bare gold as well as the kinetics of loss of HS-C 6 -ssDNA from the surface during subsequent treatment with mercaptohexanol can be understood in terms of a simple physical model and self-consistent parameters. The kinetics of HS-C 6 -ssDNA adsorption on bare gold are compared to the kinetics of hybridization of surfaceattached thiolated ssDNA with the fully complementary ssDNA in free solution and found to follow remarkably similar kinetic pathways. In contrast, the adsorption of ssDNA follows complex kinetics that cannot be modeled with a single kinetic step. That is, the presence of a thiol functionality on a 25-mer ssDNA gives rise to adsorption behavior that is clearly kinetically distinct from simple ssDNA adsorption on gold.