The main objective of this work was to evaluate the influence of incubating conditions on the specific methanogenic activity (SMA) test in order to harmonize the test protocol. For this serum bottles were incubated with anaerobic sludge (from UASB reactor treating domestic sewage) in factorial planned experiments which assessed the influence of the temperature, substrate concentration, food/microorganism (F/M) ratio, presence of yeast extract in the medium, as well as type of carbon and nutrient solution. The results showed that the tested methane measuring methods (volumetric with biogas characterization, volumetric with gas wash in alkaline solution, and manometric by using the OxiTop system) presented a similar performance. The maintenance of a small gaseous phase volume (e.g. 10% of the total volume) resulted in higher SMA values; and the ideal substrate concentration for the SMA test ranged from 0.5 to 3.0 g COD/l since higher acetate concentration caused sludge inhibition. The suggested temperature for the test is 35 degrees C and the best F/M ratio varied from 0.125 to 0.750 g COD/g VS, and this seemed to be the most influent parameter for the SMA test. Finally tests performed with nutrient solution complemented by yeast extract resulted in the highest SMA values.