Abstract The spatial distribution of microbial biomass and microbial activity was studied in two Swedish railway embankments (located close to Mora and Nässjö). Basal respiration and substrate‐induced respiration (SIR) as well as the active ( r ) and dormant ( K ) fractions of the microbial biomass were determined in samples taken from the surface layer of the ballast. The SIR, r , K and basal respiration values were all of an overall low magnitude, but were highly variable over short distances on the track. Both the biomass and basal respiration were higher on the sides of the embankment than in the middle of the tracks. SIR, K and basal respiration were correlated to the organic matter content as measured by loss on ignition, whereas r was not. None of the parameters correlated with pH. It is proposed that organic matter content could be used as a predictor for microbial biomass in railway embankments. Copyright © 2004 Society of Chemical Industry