Metabolic transformation of highly hydrophobic organic chemicals (HOCs) is one of the most important factors modulating their persistence, bioaccumulation and toxicity. Although sorption of HOCs to cellular matrices affects their bioavailability, it is still not clear how the cellular binding or sorption of HOCs in in vitro metabolism assays influences their enzymatic transformation kinetics. To elucidate effects of non-specific binding to enzymes, we measured apparent enzyme kinetics in an in vitro assay using four polycyclic aromatic hydrocarbons (phenanthrene, anthracene, pyrene and benzo[a]pyrene) as model HOCs and S9 mixture isolated from rat liver as a model enzyme mixture. The effects were also investigated in the presence of bovine serum albumin (BSA), which served to isolate the effect of protein binding from transformation. The observed transformation rates were much higher than those predicted assuming that only freely dissolved HOCs are available for metabolism. A new model including kinetic exchanges between non-specifically bound HOCs and those bound to active enzyme binding sites explained the apparent transformation kinetics at various experimental conditions better. The results are relevant for in vitro-in vivo extrapolation because the metabolic transformation rate in vivo may depend strongly on the local enzyme density and the micro-cellular environment. While non-specific protein binding reduces the unbound fraction of chemicals, this effect could be partially compensated by the facilitated transport to the active sites of the enzymes.