Dye switching and biological replication substantially increase the cost and the complexity of cDNA microarray studies. The objective of the present analysis was to quantitatively assess the importance of these procedures to provide a quantitative basis for decision-making in the design of microarray experiments. Taking advantage of the unique characteristics of a published data set, the impact of these procedures on the reliability of microarray results was calculated. Adding a second microarray with dye switching substantially increased the correlation coefficient between observed and predicted ln(ratio) values from 0.38 +/- 0.06 to 0.62 +/- 0.04 (n = 12) and the outlier concordance from 21 +/- 3% to 43 +/- 4%. It also increased the correlation with the entire set of microarrays from 0.60 +/- 0.04 to 0.79 +/- 0.04 and the outlier concordance from 31 +/- 6% to 58 +/- 5% and tended to improve the correlation with Northern blot results. Adding a second microarray to include biological replication also improved the performance of these indices but often to a lesser degree. Inclusion of both procedures in the second microarray substantially improved the consistency with the entire set of microarrays but had minimal effect on the consistency with predicted results. Analysis of another data set generated using a different cDNA labeling method also supported a significant impact of dye switching. In conclusion, both dye switching and biological replication substantially increased the reliability of microarray results, with dye switching likely having even greater benefits. Recommendations regarding the use of these procedures were proposed.