The different oxygen affinities of ammonia-oxidizing (AOB) and nitrite-oxidizing bacteria (NOB) are often used to define the operational strategy to achieve partial nitritation (PN) required before the anammox (AMX) process. For this purpose, apparent kinetic parameters are mainly used in the case of granular sludge, which can lead to errors when defining the operational conditions to obtain only nitritation (avoiding nitratation). In the present study, a mathematical methodology is proposed to determine the intrinsic kinetic parameters of AOB and NOB in granular sludge based on data obtained by respirometric assays. Additionally, the oxygen affinity constant (KO2) and maximum specific rate (rmax) of flocculent and granular sludge sample, produced under mainstream and sidestream conditions were determined at various temperatures (15, 20 and 30 °C). The results show that for granules, the intrinsic KO2 and rmax values were lower and higher, respectively, than the apparent values. Furthermore, the KO2 values for flocs and granules at all of the tested temperatures were lower for NOB than for AOB. The values obtained for the kinetic parameters indicated that it is impossible to maintain partial nitritation by only controlling the dissolved oxygen concentration.