This article describes a novel microfluidic 3-dimensional encapsulation method via the self-assembling peptide hydrogel. The microfluidic immobilization strategies using a peptide hydrogel have been designed for microfluidic cell-based assays, cocultures, and biomimetic micro blood vessels. A sol-gel transition peptide hydrogel, Puramatrix, is adopted for use in the microfluidic device fabricated by photolithography and a poly(dimethylsiloxane) replica molding process. The peptide hydrogel was hydrodynamically focused by sheath flows of distilled water and cell culture media, and gelled by diffusion of media. After being transitioned from a sol to gel phase, the fabricated scaffold in the middle of the main channel was not washed away via fluid flows. The diffused chemicals in a stripe-shaped peptide scaffold of microchannel formed a linear concentration gradient within the scaffold. Based on application in an in vivo-like 3-dimensional microenvironment, this microfluidic system could be applied to cocultures, angiological research, cytotoxicity tests, cell viability monitoring, and continuous dose-response assays as well as drug-drug interaction studies. ? 2006 The Association for Laboratory Automation.