In this report, we describe the construction and analysis of a cell-free protein synthesis system immobilized in calcium alginate microbeads. When incubated in a feeding solution that contained amino acids and other low-molecular-weight substrates, the microbeads transcribed and translated coimmobilized DNA into functional proteins. Protein synthesis continued for more than 15 h with the diffusional supply of substrates and removal of by-products. In addition, functional proteins were generated from PCR-amplified genes as efficiently as from plasmid, suggesting that these cell-like microbeads could be used for functional screening of genomic libraries.