This work describes the loading capacity of poly(D,L-lactide) nanoparticles, the factors influencing pentamidine release, and the cytotoxicity of nanoparticles. The nanoprecipitation method was used to prepare pentamidine-loaded poly(D,L-lactide) nanoparticles. Various concentrations of pentamidine base and polymer were tested. The influence of the dilution, temperature, and ionic strength was evaluated. The cytotoxicity on J 774 cells of unloaded nanoparticles, pentamidine-loaded nanoparticles, and pentamidine isethionate were tested. The percentage of binding decreased significantly with drug load. A nonlinear increase in drug uptake per unit mass of polymer with the equilibrium pentamidine concentration was found. A Langmuir-type sorption was suggested (r = 0.998). 390 µg/ml was found to be the highest level of drug incorporation. The increase of polymer concentration did not improve the pentamidine fixation yield. The increase in temperature or buffer molarity induced a significant release of pentamidine. The increase in dilution also induced an increase in release of pentamidine. The cytotoxicity of pentamidine-loaded nanoparticles and unloaded nanoparticles was superimposable. After 24 h of incubation, pentamidine-loaded nanoparticles presented an IC 50 value significantly lower than that of free drug (0.39 vs. 6.5 g/ml).