X-ray diffraction (XRD) provides important information regarding a three-dimensional molecular structure, but high-quality crystals are necessary for XRD analysis. However, it is often very difficult to obtain high-quality protein crystals. We previously found that protein crystals can be forcibly generated by femtosecond laser irradiation and have succeeded in crystallizing soluble and membrane proteins. The mechanism of this phenomenon is still unknown. In this study, we examined the relation between the probabilities of nucleation and crystallization conditions by femtosecond laser irradiation, and were able to induce nucleation by laser irradiation under solution conditions in the metastable region where crystals do not form spontaneously. The induction efficiency increased as the conditions approached those of the spontaneous nucleation area. The result suggests that protein concentrations are changed by laser irradiation. We subsequently performed high-speed imaging of cavitation bubbles generated by laser irradiation to reveal the role of cavitation in nucleation. A ring of highly concentrated protein solution was observed around the cavitation. These results indicate that the cavitation bubble produced by laser irradiation creates temporary local protein high-concentration regions, which could be the trigger for nucleation. (C) 2010 Elsevier B.V. All rights reserved.