Pharmaceuticals are becoming increasingly prevalent in the environment and regulations to assess the environmental risks requires the use of fish in animal testing. A primary fish gill cell culture system has shown significant promise as an in vitro replacement model system for whole fish compound uptake studies. The current study assessed the uptake of 8 pharmaceuticals with a range of chemical parameters from the apical freshwater compartment of the primary gill cell culture system. An HPLC method was developed to extract and analyze compounds simultaneously from the OECD water matrix (warfarin, ibuprofen, beclomethasone, carbamazepine, diclofenac, ketoprofen, norethindrone, gemfibrozil). The relationship between the uptake of the compounds and the various checmial parameters were analysed using linear regression. There was no correlation to Log Kow (R=0.002), weaker correlation to Log S (R2=0.24) and molecular weight (R2=0.23), and a stronger correlation to Log D (R2=0.57), and pKa (R2=0.56). The strength of correlation of Log S (R2=0.83) and pKa (R2=0.91) were greatly improved with carbamazepine and diclofenac excluded, respectively. Uptake of the compounds was found to be a function of cell permeability and overall findings suggest that ionizable pharmaceutical are able to interact and enter fish gill cells.