Conventional MIC testing of amphotericin B results in narrow MIC ranges challenging the detection of resistant strains. In order to discern amphotericin B pharmacodynamics, the in vitro activity of amphotericin B was studied against Aspergillus isolates with the same MICs by using a new in vitro pharmacokinetic/pharmacodynamic (PK/PD) model that simulates amphotericin B human plasma levels. Clinical isolates of Aspergillus fumigatus, A. terreus, and A. flavus with the same Clinical and Laboratory Standards Institute modal MICs of 1 mg/liter were exposed to amphotericin B concentrations following the plasma concentration-time profile after single-bolus administration with C(max) values of 0.6, 1.2, 2.4, and 4.8 mg/liter. Fungal growth was monitored for up to 72 h based on galactomannan production. Complete growth inhibition was observed only against A. fumigatus with amphotericin B with a Cmax of ≥ 2.4 mg/liter. At the lower C(max) values 0.6 and 1.2 mg/liter, significant growth delays of 34 and 52 h were observed, respectively (P < 0.001). For A. flavus, there was no complete inhibition but a progressive growth delay of 1 to 50 h at an amphotericin B C(max) of 0.6 to 4.8 mg/liter (P < 0.001). For A. terreus, the growth delay was modest (up to 8 h) at all C(max)s (P < 0.05). The C(max) (95% confidence interval) associated with 50% activity for A. fumigatus was 0.60 (0.49 to 0.72) mg/liter, which was significantly lower than for A. flavus 3.06 (2.46 to 3.80) mg/liter and for A. terreus 7.90 (5.20 to 12.29) mg/liter (P < 0.001). A differential in vitro activity of amphotericin B was found among Aspergillus species despite the same MIC in the order A. fumigatus > A. flavus > A. terreus in the in vitro PK/PD model, possibly reflecting the different concentration- and time-dependent inhibitory/killing activities amphotericin B exerted against these species.