We present a method for generating gel-based unordered 2D arrays of bacterial cells of a very high density, up to 105 cells per mm2. Bacteria in a suspension are focused into a thin layer when the suspension and a dry gel matrix penetrate each other. Formation of a second gel from gel-forming components contained in the suspension results in immobilization of the cells. The immobilized cells stay alive and can repeatedly divide to produce microcolonies. The method provides for high-throughput screening and massively parallel analysis of individual cells in large populations, as well as for rapid isolation of rare clones. Biotechnol. Bioeng. 2011;108: 2682–2690. © 2011 Wiley Periodicals, Inc.