Kushiro Mire is the largest mire in Japan and in 1980 was the first wetland in Japan registered under the Ramsar Convention. Recent reports indicate an increase in nutrient loading into Kushiro Mire from changes in land use. We measured vertical profiles of dissolved inorganic nitrogen (DIN; NO(3) (-), NO(2) (-), NH(4) (+)), dissolved organic carbon (DOC), and various types of microbial activity in soil samples collected to approximately 1.5 m deep at two sites in Kushiro Mire. We found an accumulation of NO(3) (-) and DOC in the deeper soil. Denitrifying activity was highest in the shallower soils and decreased drastically with depth, whereas higher levels of fluoresceindiacetate hydrolysis, beta-glucosidase, acid phosphatase, and xylosidase enzyme activity were found in the deeper layers. We also detected humic-like substances as components of the DOC. These results suggest that the DOC in the wetland soil cannot be used as a substrate for denitrification, causing denitrification to be suppressed in the deeper soil. In addition, denitrifying activity would be very low in the deeper layers due to low soil temperature. As a result, nitrogen input to the mire has resulted in a large accumulation of NO(3) (-) in the deeper soil. This will eventually change the mire ecosystem through effects such as increased eutrophication and acidification.