The radiolabeled leucine incorporation technique for quantifying rates of bacterial production has increased in popularity since its original description for bacterioplankton communities. Prior studies addressing incorporation conditions (e.g., substrate saturation) for bacterial communities in other habitats, such as decaying plant litter, have reported a wide range of final leucine concentrations (400 nM to 50 microM) required to achieve saturation-level uptake. We assessed the application of the [(3)H]leucine incorporation procedure for measuring bacterial production on decaying wetland plant litter. Substrate saturation experiments (nine concentrations, 10 nM to 50 microM final leucine concentration) were conducted on three dates for microbial communities colonizing the submerged litter of three emergent plant species (Typha angustifolia, Schoenoplectus validus, and Phragmites australis). A modified [(3)H]leucine protocol was developed by coupling previously described incubation and alkaline extraction protocols with microdialysis (500 molecular weight cutoff membrane) of the final radiolabeled protein extract. The incorporation of [(3)H]leucine into protein exhibited a biphasic saturation curve, with lower apparent K(m) values ranging from 400 nM to 4.2 microM depending on the plant species studied. Upper apparent K(m) values ranged from 1.3 to 59 microM. These results suggest differential uptake by litter-associated microbial assemblages, with the lower apparent K(m) values possibly representing bacterial uptake and higher apparent K(m) values representing a combination of both bacterial and nonbacterial (e.g., eukaryotic) uptake.