Manual manipulation of protein crystals is often required in order to obtain X-ray diffraction (XRD) data, but the success of the manual operation depends on the experience and fortuity of the operators. Here, we demonstrated the processing of protein crystals grown in semi-solid agarose gel using a femtosecond laser. This high-precision, reproducible processing could be achieved without unsealing the crystallization trays by using a focused femtosecond laser. We confirmed that the gel-immobilized crystals of hen egg white lysozyme, glucose isomerase and thaumatin could be processed by this technique. In contrast, the processing of protein crystals grown in non-gelled solution triggered polycrystallization or was unsuccessful. The processed gel-grown lysozyme crystal was subsequently captured by a nylon loop without difficulty and mounted onto the goniometer head of the XRD equipment for XRD data collection. The statistics of the obtained XRD data indicated that laser irradiation has little influence on crystallinity, suggesting that the processed protein crystals are virtually suitable for X-ray analysis. This approach provides a reliable method of processing protein crystals and may lead to an automated system for protein crystal processing. (C) 2009 Elsevier B.V. All rights reserved.