Carbon content and indices of microbial biomass and activity were determined in 1985 in different soil horizons of two nitrogen-fertilized pine forests in Sweden. The Kroksbo site was fertilized in 1974 with 150 and 600 kg N•ha−1 using NH4NO3 or urea, while the Nissafors site was fertilized in 1977 and 1984 with 150 kg NH4NO3-N•ha−1 The absolute amount of C per square metre of forest floor increased in fertilizer treatments compared with the control (by 10–26%, depending on the site or fertilizer treatment). Respiration rate, ATP content, and microbial biomass C, measured with the substrate-induced respiration technique, decreased in all horizons when expressed per gram of C. The decrease was most evident with NH4NO3, and at the highest level of fertilization. However, on an area basis there were no differences between fertilizer and control treatments. A calculated increase in litter fall in the fertilized plots could not explain all the increase in the amount of C per square metre compared with the control. Decreased microbial activity per gram of C therefore appeared to be an important factor in the increase in C content in fertilized plots.