Tingting Wu and Mark Coyne, Dept. of  Plant and Soil Sciences, N122 Ag Science North Univ of Kentucky, Lexingtion, KY 40546-0091 Severe limitations have been recognized in culture-based techniques for assessing denitrifier populations. Alternately, functional genes involved in denitrification have been extensively and effectively used to study the highly diverse denitrifying microbes in various environments. In our study, denitrifier community was evaluated in soils with identified fragipan, the environment in which stratified denitrifier population from surface to the depth of fragipan were found via traditional cultural method.  Primers nirS and nirK, encoding nitrite reductase, which catalyzes the rate-limiting step in denitrification, were used for the PCR amplification of DNA extract from various soils by depth. The Cu-type nitrite reductase NirK  was found in both surface and subsurface soils while heme-type NirS enzyme was only found in the surface samples, indicating that different denitrifier community structures occurred at different depth of soils. Dentrifier population numbers indicated by most probable number analysis were so low (< 400 MPN g^-1 soil) that we failed to extract DNA only using common commercial kit at the fragipan boundary layer. However, we found that skim milk could greatly improve DNA recovery from our subsurface soil samples and provide useable DNA fro PCR amplification. Our results of the nitrite reductase gene analysis suggested that the community structure of denitrifiers differs by depth in these soil environments.