We have developed a class of peptide amphiphile (PA) molecules that self-assemble into three-dimensional nanofiber networks under physiological conditions in the presence of polyvalent metal ions. The assembly can be triggered by adding PA solutions to cell culture media or other synthetic physiological fluids containing polyvalent metal ions. When the fluids contain suspended cells, PA self-assembly entraps cells in the nanofibrillar matrix, and the cells survive in culture for at least three weeks. We also show that entrapment does not arrest cell proliferation and motility. Biochemical and ultrastructural analysis by electron microscopy indicate that entrapped cells internalize the nanofibers and possibly utilize PA molecules in their metabolic pathways. These results demonstrate that PA nanofibrillar matrices have the potential to be used for cell transplantation or other tissue engineering applications.