A possible artefact ofin situmeasurements of nitrogen (N) net mineralization may result from the inclusion of abscised fine roots in incubated soil. Fine roots usually have a relatively wide carbon (C)/N ratio and if fine root carbon is available to micro‐organisms they will immobilize N. In this study nitrogen and carbon in microbial biomass were compared between incubated soil and surrounding soil at the end of field incubation periods. It was hypothesized that immobilization of N would result in higher microbial N in incubated soil when compared to the surrounding soil. Incubations were carried out in steel tubes at sites of different fine root density. No net immobilization of N occurred during incubations. There were no significant increases of microbial N in incubated soil suggesting that immobilization of N in microbial biomass did not occur. A comparison of K2SO4‐extractable organic N concentrations between incubated and surrounding soil also indicated that immobilization of N into extractable metabolites was unlikely. However, under herbaceous vegetation significantly lower microbial N concentrations were measured in incubated soil compared to the surrounding soils. This may suggest a decrease in microbial biomass during incubations due to cessation of C input from fine roots. If the reduction in microbial biomass leads to the mineralization of microbial tissue, net N mineralization using thein situincubation technique may be overestimated.