The measurement of ATP as an estimate of microbial biomass C in humus was studied. Three different acid extractants, H2SO4, TCA and H3PO4 were compared, as well as two different buffers, Tris and arsenate. No difference in extraction efficiency was found when the light output was converted to ATP content using internal ATP standards, but the different extraction agents and buffers influenced the light output. Several pretreatments and storage conditions were investigated. Incubation at 25°C for 1–7 days gave ca 50% higher ATP values than incubation at 4°C. Sieving did not affect the result. For long-term storage of humus, 4°C is recommended instead of freezing, since freezing reduced the ATP content to ca 80% of the amount found in humus stored at 4°C after 2 months. Freezing of the humus extract resulted in even larger losses of ATP. Humus from eight coniferous forests in Sweden was investigated with respect to ATP content and microbial biomass, estimated with the substrate induced respiration rate method (SIR). A close linear relationship (r = 0.95) was found between the two types of measurement. Calculated from this relationship the amount of ATP (at 25°C) was 3.2μmg−1 biomass C.