AbstractWe have measured single‐cell gene expression over time using a microfluidics‐based flow cell which physically traps individual yeast using µm‐sized structures (yeast jails). Our goal was to determine variability of gene expression within a cell over time, as well as variability between individual cells. In our flow cell system, yeast jails are fabricated out of PDMS and gene expression is visualized using fluorescently‐tagged proteins of interest. Previously, single‐cell yeast work has been done using micromanipulation on agar, or FACS. In the present device agar is eliminated, resulting in a superior optical system. The flow of media through the flow cell washes daughter cells away, eliminating the need for micromanipulation. Unlike FACS, the described device can track individual yeast over a time course of many hours. The flow cells are compatible with the needs of quantitative fluorescence microscopy, and allow simultaneous measurements to be done on a large number of individual yeast. We used these flow cells to determine the expression of HSP104‐GFPand RAS2‐YFP, genes known to affect yeast life span. The results demonstrate inter‐cell variation in expression of both genes that could not have been detected without this single‐cell analysis. Copyright © 2006 John Wiley & Sons, Ltd.