Chemostat cultivation of micro-organisms offers unique opportunities for experimental manipulation of individual environmental parameters at a fixed, controllable specific growth rate. Chemostat cultivation was originally developed as a tool to study quantitative aspects of microbial growth and metabolism. Renewed interest in this cultivation method is stimulated by the availability of high-information-density techniques for systemic analysis of microbial cultures, which require high reproducibility and careful experimental design. Genome-wide analysis of transcript levels with DNA micro-arrays is currently the most commonly applied of these high-information-density analysis tools for microbial gene expression. Based on published studies on the yeast Saccharomyces cerevisiae, a critical overview is presented of the possibilities and pitfalls associated with the combination of chemostat cultivation and transcriptome analysis with DNA micro-arrays. After a brief introduction to chemostat cultivation and micro-array analysis, key aspects of experimental design of chemostat-based micro-array experiments are discussed. The main focus of this review is on key biological concepts that can be accessed by chemostat-based micro-array analysis. These include effects of specific growth rate on transcriptional regulation, context-dependency of transcriptional responses, correlations between transcript profiles and contribution of the corresponding proteins to cellular function and fitness, and the analysis and application of evolutionary adaptation during prolonged chemostat cultivation. It is concluded that, notwithstanding the incompatibility of chemostat cultivation with high-throughput analysis, integration of chemostat cultivation with micro-array analysis and other high-information-density analytical approaches (e.g. proteomics and metabolomics techniques) offers unique advantages in terms of reproducibility and experimental design in comparison with standard batch cultivation systems. Therefore, chemostat cultivation and derived methods for controlled cultivation of micro-organisms are anticipated to become increasingly important in microbial physiology and systems biology.