This study aimed to comprehensively investigate the in vitro metabolism of statins. The metabolism of clinically relevant concentrations of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin and their metabolites were investigated using human liver microsomes (HLMs), intestine microsomes (HIMs), liver cytosol, and recombinant cytochrome P450 (CYP) enzymes. We also determined the inhibitory effects of statin acids on their pharmacological target, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. In HLMs, statin lactones were metabolized to a much higher extent than their acid forms. Atorvastatin lactone and simvastatin (lactone) showed extensive metabolism (intrinsic clearance (CL int ) values of 3,700 and 7,400 µl/min/mg), while the metabolism of the lactones of 2-hydroxyatorvastatin, 4-hydroxyatorvastatin, and pitavastatin was slower (CL int 20-840 µl/min/mg). The acids had CL int values in the range <0.1-80 µl/min/mg. In HIMs, only atorvastatin lactone and simvastatin (lactone) exhibited notable metabolism, with CL int values corresponding to 20% of those observed in HLMs. CYP3A4/5 and CYP2C9 were the main statin-metabolizing enzymes. The majority of the acids inhibited HMG-CoA reductase with 50% inhibitory concentrations of 4-20 nM. The present comparison of the metabolism and pharmacodynamics of the various statins using identical methods provides a strong basis for further application, e.g., comparative systems pharmacology modelling.