Recent work in our laboratory indicated that the slow rate of denitrification in Iowa subsoils is not due to a lack of denitrifying microorganisms, but to a lack of organic C that can be utilized by these microorganisms for reduction of nitrate. To identify factors affecting the availability of leachable organic C in surface soils capable of promoting denitrification in subsoils, we studied the effects of freezing and drying and of plants and plant residues on the amounts of water-soluble organic C in surface soils and the ability of this organic C to promote denitrification in subsoils. We found that aqueous extracts of field-moist, frozen, and air-dried surface soils promoted denitrification in subsoils and that their stimulatory effects on denitrification were highly correlated (r=0.93) with their organic C contents and decreased in the order air-dried soils ≫ frozen soils >field-moist soils. But a detailed study of the effect of drying a surface soil to different water tensions indicated that drying of soils under natural conditions is not likely to lead to a substantial increase in their content of water-soluble organic C. Amendment of surface soils with corn or soybean residues led to a marked increase in the amount of organic C in aqueous extracts of the soils and in the ability of these extracts to promote denitrification in subsoils. These effects of plant residues could not be detected after incubation of residue-treated soils for a few days under aerobic conditions, but they increased markedly with an increase in the time of incubation from 1 to 10 days when residue-treated soils were incubated under anaerobic conditions. Analyses for organic acids indicated that this increase was largely due to fermentative production of acetic, propionic, and butyric acids by soil microorganisms. Growth chamber studies showed that growth of corn, soybean, wheat, and sorghum plants on surface soil did not significantly increase the organic C content of leachates of the soil or the ability of these leachates to promote denitrification in subsois. We conclude that plant residues are a major source of the leachable organic C in surface soils that is capable of promoting denitrification in subsoils.