A functional gene microarray was used to investigate denitrifier community composition and nitrite reductase (nirS) gene expression in sediments along the estuarine gradient in Chesapeake Bay, USA. The nirS oligonucleotide probe set was designed to represent a sequence database containing 539 Chesapeake Bay clones, as well as sequences from many other environments. Greatest nirS diversity was detected at the freshwater station at the head of the bay and least diversity at the higher salinity station near the mouth of the Bay. The most common OTUs from the sequence database were detected on the array with high signal strength in most samples. One of the most abundant OTUs, CB2-S-138, was identified as dominant at the mid-bay site by both microarray and quantitative PCR assays, but it comprised a much smaller fraction of the assemblage in the north and south bay samples. cDNA (transcribed from total RNA extracts) targets were hybridized to the same array to compare the profiles of community composition at the DNA (relative abundance) and mRNA (gene expression) levels. Only the three dominant denitrifying groups (in terms of relative strength of DNA hybridization signal) were detected at the mRNA level. These results suggest that the most actively denitrifying groups are responsible for most nirS expression as well.