Development of the techniques for fabricating three-dimensional tissues still poses significant challenges for tissue engineering. We used hydrogels obtained from phenol-substituted amylopectin (AP-Ph) as templates for preparing multicellular spherical tissues (MSTs) and endothelialized curved tubular structures in type I collagen gel. AP-Ph hydrogel microparticles of diameter 200?mu m and fibers of diameter 500?mu m disappeared within hours of soaking in a serum-containing medium. HeLa cells and human endothelial cells were enclosed in the microparticles and hydrogel fibers, respectively, and then embedded in Ca-alginate microcapsules or the collagen gel. The enclosed cells were released in cavities formed by hydrogel degradation in the serum-containing medium. The released HeLa cells in the spherical cavities grew and formed MSTs, eventually filling the cavities. The spherical tissues were easily harvested by liquefying the Ca-alginate hydrogel microcapsule membrane by chelation using sodium citrate. The released endothelial cells grew on the tubular cavity surfaces and formed tubular structures. An endothelial cell network was formed by cell migration into the collagen gel. These results demonstrate the potential of serum-degradable AP-Ph hydrogels in constructing three-dimensional tissues. Biotechnol. Bioeng. 2012; 109: 29112919. (c) 2012 Wiley Periodicals, Inc.