Proper activation of Fe catalysts is an important step in determining their activity for the Fischer-Tropsch synthesis (FTS). The results of this study reveal for the first time the effect of activation and time on stream (TOS) on intrinsic site activity and concentration of surface intermediates during CO hydrogenation (methanation) on an Fe FTS catalyst. The catalyst was activated under identical conditions but with different pretreatment gases: CO ([CO]), H-2 ([H]), or syngas ([S]). Lifetime (tau(M)) and concentration of methane surface intermediates (N-M) were measured in situ using isotopic tracing (SSITKA) of CO hydrogenation under methanation conditions (H-2 : CO = 10: 1, T = 280 degreesC, P = 1.8 atm). Fe phases after activation were found by XRD to be Fe-0 + Fe3O4 for [H] and Fe carbides + Fe3O4 for both [CO] and [S]. Reaction and SSITKA results showed that the rate and abundance of surface intermediates on the [H]-pretreated catalyst developed with TOS, reaching a maximum at ca. 1 h, and then declined to steady-state values at 21 h, still significantly higher than for the other pretreated samples. Activity was shown by SSITKA to be primarily determined by the number of active intermediates (related to the number of surface sites). Measures (1/tau(M)) of the intrinsic site activity on the differently activated catalyst samples were not significantly different, suggesting that the active sites were all identical. Given the similarity in the activity of the sites and the increase in the concentration of active sites (and rate) of [H] and [CO] during the initial reaction period, it can be concluded that the active sites are probably on a (partially?) carburized Fe surface.