Nanocrystal technology is one of a promising approach used to improve the solubility of poorly soluble drugs. In this study, media milling technique was used to produce clarithromycin nanocrystals via a bead milling machine. Various sizes of clarithromycin nanocrystals (250 nm-1 mu m) were prepared using different milling times. The polymorphism and crystallinity of nanocrystals were characterized by powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC) techniques. The results indicated no polymorphic change after the milling process. However, the crystallinity of the obtained nanocrystals slightly decreased upon the milling time. The kinetic saturation solubility of all sizes of the clarithromycin nanocrystals was found to reach a maximum solubility or the supersaturation within an hour in HBSS buffers pH 6.8 and 5.0. The maximum solubility of the smaller nanocrystals was higher than that of the larger ones. In addition, the dissolution rate of the nanocrystals was enhanced after the particle size reduction. Permeability and cellular uptake of the clarithromycin nanocrystals were observed in Caco-2 and NCI-N87 cells. No significant difference in drug permeation of the different size nanocrystals was observed in NCI-N87 cell monolayer due to the high solubility of clarithromycin in an acidic environment. However, the drug transport, the apparent permeability coefficient (P-app) and the drug internalization in Caco-2 cell monolayer were found to increase when the particle size of clarithromycin was reduced to the nanosized range. Based on the results, the particle size strongly influenced the saturation solubility, dissolution rate, in vitro drug permeation and internalization of clarithromycin nanocrystals. (C) 2018 Elsevier B.V. All rights reserved.