The European Commission recognises the essential role of soil biology in soil functioning and delivery of ecosystem services, but information is currently lacking evaluate of how these vary across soil and land-use types at a European scale. This study evaluated the measurement of the initial rate of soil basal respiration (BR) as a potential biological indicator of ecosystem service provision. The purpose of this study was to test ISO 16072:2002 (Soil Quality: Laboratory methods for the determining of microbial soil respiration). In the literature a range of pre-incubation temperatures (pre-inc) and experimental incubation temperatures (exp-inc) have been applied when using the ISO method for the establishment of basal respiration. This study evaluated whether the range of temperatures applied during pre- and exp- incubation had a significant effect on the rate of respiration determined when following the protocol established in ISO 16072:2002. The evaluation was carried out on a pedo-climatic gradient spanning ten countries across Europe and covering four biogeographical regions. Three sites were sampled in each country providing a range of soil and land-use parameters. Our results suggest that experimental incubation temperatures of 20 ◦C or above should be used in the application of the methodology ISO 16072:2002 (incubation at 15 ◦C resulted in erratic variation between replicates). However, pre-incubation temperature did not affect the soil basal respiration rate, when following the standard recommendations. The time interval with the best prediction of the initial rate of basal respiration was 6 h. ; ENVironmental ASsessment of Soil for mOnitoring(ENVASSO) Project, funded as Scientific Support to Policy (SSP) under the European Commission 6th Framework Programme (Con-tract 022713, 2006–8).