Milling and supercritical fluid technology have been used to prepare particles suitable for pulmonary delivery, but problems remain, e.g., the loss of protein activity, the heterogeneity of drug particles including shape, size etc., process yield, and the applicability for industrial production process. Therefore, in order to develop a novel process for particle preparation, crystallization was investigated using insulin as a model protein. The relation between the number of seed particles and the final crystal size was investigated, and a novel microcrystallization process, named "seed zone" method, was developed. The size of crystals was inversely proportional to the number of seeds in crystallizing solution. Spontaneous crystallization occurred around pH 6 in acetic acid solution, however, more than 60% of crystals were bigger than 5 mum with two peak size distributions. On the contrary, microcrystals with a mean diameter of 3 mum were prepared using a seed zone method. The "seed zone" is a pH range where the seed particles are stable in crystallizing solution. Almost 90% (in volume) of microcrystals were under 5 gm, and the yield of crystallization was maintained at 90% or higher. In the seed zone, nano-sized particles (96 nm) with narrow size distributions were identified. Therefore, it is likely that these nano-sized particles would be used as seeds in microcrystals formation. It is suggested that insulin microcrystallization using a seed zone could be a useful particle preparation process in pharmaceutical industry. (C) 2003 Elsevier B.V. All rights reserved.