A new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200-to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis ⌬fur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwater system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a highthroughput fashion.Microarray-based genomic technology is a powerful tool for viewing the expression of thousands of genes simultaneously in a single experiment (13). While this technology was initially designed for transcriptional profiling of a single species, its applications have been dramatically extended to environmental applications in recent years (1,3,4,7,8,20,23,30,32,33,46,47,48). One of the greatest challenges in using microarrays for analyzing environmental samples is the low detection sensitivity of microarray-based hybridization in combination with the low biomass often present in samples from environmental settings. We previously developed a DNA-based microarray detection approach coupled with whole-community genome amplification, and we utilized this technique to analyze microbial community structure and demonstrated that it can be used for low-biomass groundwater microbial communities (41). However, this approach could not be directly adapted and used for mRNA-based activity analyses.A practical problem in detecting mRNAs from environmental samples by microarray hybridization is obtaining a sufficient amount of mRNAs for analysis. Some type of signal amplification prior to hybridization is needed. However, random PCR-based amplification is not an appropriate choice due to amplification bias and thus the loss of ...