Virus-like particles (VLP) have received considerable attention for vaccine, drug delivery, gene therapy and material science applications. Although the number of unique VLP and their applications are rapidly growing, the positive impact of VLP applications is limited by the current diverse, expensive, and typically low-yielding production technologies available. These technologies, when scaled, often result in structurally and compositionally inconsistent products. We present Escherichia coli-based cell-free protein synthesis as a production technology to overcome many of the limitations of current VLP production processes. Using this technique, the MS2 bacteriophage coat protein VLP was produced at a yield 14 times the best published production yield. Also, a C-terminally truncated Hepatitis B core protein VLP was produced at similarly high yields (6 × 1013 VLP/mL). These VLP were found to have comparable characteristics to those produced in vivo. The scalability of this technology was tested without loss in production yields. To our knowledge, this is the first time a prokaryote-based in vitro transcription/translation system has generated a virus-like particle. Biotechnol. Bioeng. 2008;100: 28–37. Biotechnol. Bioeng. 2008;100: 28–37. © 2007 Wiley Periodicals, Inc.