Mycophenolate mofetil is used in rejection prophylaxis after allograft transplantation. The highly variable pharmacokinetics and pharmacodynamics (PD) of the active moiety mycophenolic acid (MPA) render this drug attractive for therapeutic monitoring. The aim of this study was to characterize the exposure-response relationship for MPA to guide future strategies for individualized therapy based on PD monitoring. A single-dose (100, 250, 500, and 1000 mg mycophenolate mofetil) crossover exposure-response study of MPA PD in CD4+ cells was performed in 5 healthy individuals. The activity of inosine 5′-monophosphate dehydrogenase (IMPDH) at time 0 ranged from 1.2 to 7.2 pmol per 106 cells/min. IMPDH was strongly inhibited by MPA; MPA EC50 (concentration required for 50% inhibition) of 2.3 mg/L was determined by a pooled data analysis. Decreased IMPDH gene expression was associated with the exposure to MPA. There were no immediate reductions of guanine nucleotides. On the contrary, a trend toward increased guanosine triphosphate was observed. IMPDH activity AUC0-12h approached maximum reduction at MPA AUC0-12h 22 mg·h/L (corresponding to the 500 mg dose), whereas plasma concentrations exceeding approximately 6 mg/L did not further increase the IMPDH inhibition. The results suggest that guanine nucleotides in circulating lymphocytes may not serve as immediate response biomarkers to MPA. Strategies for preventing over- or underexposure to MPA may be developed by means of IMPDH activity combined with MPA concentration measurement.