Although plasma protein binding (PPB) is accepted to be an essential factor in reducing antimicrobial activity, little is known about the underlying mechanisms. One possibility includes impaired penetration of an antimicrobial into bacterial cells in the presence of PPB. As a prerequisite for testing this hypothesis an optimized medium displaying high protein binding without impairing bacterial growth had to be identified for our model compound clindamycin. Determination of PPB, bacterial growth and antimicrobial killing was performed in Mueller-Hinton broth (MHB) containing various amounts of human albumin or serum. [(3)H]clindamycin was used to investigate clindamycin penetration into Staphylococcus aureus. Of all investigated media only MHB(50%serum) and MHB(70%serum) achieved protein binding comparable to pure serum. In contrast, MHB(20%serum) and most media containing only albumin demonstrated considerably lower protein binding. Pure serum resulted in bacterial growth inhibition compared with MHB while MHB(16%albumin) and MHB(50%serum) did not result in significant differences in bacterial count after 24 h. However, in both MHB(16%albumin) and MHB(50%serum) the antimicrobial activity of clindamycin was reduced by > 2 log(10) cfu/mL compared with pure MHB. The radioactive signal after administration of [(3)H]clindamycin to S. aureus was significantly decreased in pure serum as well as in MHB(16%albumin) and MHB(50%serum), while no significant difference was observed for MHB(4%albumin) and MHB(20%serum). Reduction of the intracellular radioactive signal in the presence of serum proteins correlated both with the degree of protein binding and reduction of antimicrobial activity supporting the hypothesis of impairment of activity by PPB by reducing intra-bacterial antimicrobial concentrations.