Vacuolated spherical kafirin microparticles with a mean diameter of 5 mu m can be formed from an acidic solution with water addition. Three-dimensional scaffolds for hard tissue repair require large structures with a high degree of interconnected porosity. Cross linking the formed kafirin microparticles using wet heat or glutaraldehyde treatment resulted in larger structures (approximately 20 mu m), which, while similar in size and external morphology, were apparently formed by further assisted assembly by two significantly different mechanisms. Heat treatment, which increased the vacuole size, involved kafirin polymerization by disulfide bonding with the microparticles being formed from round, coalesced nanostructures, as shown by atomic force microscopy (AFM). Kafirin polymerization, of glutaraldehyde-treated microparticles was not by disulfide bonding, and the nanostructures, as revealed by AFM, were spindle shaped. Both treatments enhanced BMP-2 binding to the microparticles, probably due to their increased size. Thus, these. Modified kafirin microparticles have potential as natural, nonanimal protein bioactive scaffolds.