Assessment of reproducibility of DNA-microarray analysis from published data sets is complicated by the use of different microbial strains, cultivation techniques, and analytical procedures. Because intra- and interlaboratory reproducibility is highly relevant for application of DNA-microarray analysis in functional genomics and metabolic engineering, we designed a set of experiments to specifically address this issue. Saccharomyces cerevisiae CEN.PK113-7D was grown under defined conditions in glucose-limited chemostats, followed by transcriptome analysis with Affymetrix Gene-Chip arrays. In each of the laboratories, three independent replicate cultures were grown aerobically as well as anaerobically. Although variations introduced by in vitro handling steps were small and unbiased, greater variation from replicate cultures underscored that, to obtain reliable information, experimental replication is essential. Under aerobic conditions, 86% of the most highly expressed yeast genes showed an average intralaboratory coefficient of variation of 0.23. This is significantly lower than previously reported for shake-flask-culture transcriptome analyses and probably reflects the strict control of growth conditions in chemostats. Using the triplicate data sets and appropriate statistical analysis, the change calls from anaerobic versus aerobic comparisons yielded an over 95% agreement between the laboratories for transcripts that changed by over 2-fold, leaving only a small fraction of genes that exhibited laboratory bias.