Many biosensor applications are based on streptavidin (SA) binding to partially biotinylated self-assembled thiol monolayers (SAMs). In our study, binary SAMs on gold were prepared from solutions containing 16-mercapto-1-hexadecanol (thiol I) and N-(8-biotinyl-3,6-dioxa-octanamidyl)-16-mercaptohexadecanamide (thiol II) in varying component ratios. Either chloroform or ethanol was used as solvent. After 24 h thiol incubation, SA was immobilized on the resulting SAMs using the strong SA-biotin interaction. The SA binding process was monitored by QCM-D (quartz crystal microbalance monitoring dissipation factor). It is shown that the Sauerbrey equation is valid to calculate the mass quantities of the immobilized SA layers. Under the chosen incubation conditions, marginal fractions of the biotinylated component II in chloroform ((n(I)/n(II))(solution) approximate to 1000) lead to SAMs which ensure a maximal SA binding quantity of m(Saurbrey SA) approximate to 400 ng.cm(-2), being equivalent to a SA single-layer arrangement on the SAM surface. In case of incubations from ethanolic solutions, a complete SA layer formation needs significantly higher amounts of the biotinylated component II during SAM preparation ((n(I)/n(II))(solution) approximate to 50). X-ray photoelectron spectroscopy data show that the fraction of biotinylated thiol II in the SAM determines the amount of surface-bound SA. The SAM thiol ratio ((n(I)/n(II))(SAM)) not only depends on the corresponding component ratio in the incubation solution, but is also strongly influenced by the solvent. Using chloroform as solvent during SAM preparation significantly increased the fraction of biotinylated thiol II in the SAMs compared to ethanol.