Population size estimates must be comparable through time to interpret trends in threatened carnivore populations. Because prey distribution and dynamics drive carnivore distribution, and sampling methods often utilise behavioural responses to attractants, habitat variability among sampling occasions may confound such estimates. We explored whether a marked and unexpected reduction in lion Panthera leo population size estimates in Kruger National Park, was real or represented changes in behavioural responses to call-up stations given reduced rainfall between sampling periods. Rainfall drives savannah landscape heterogeneity, surface water and lion prey food availability. Hence landscape factors should more strongly influence lion behaviour during dryer conditions. We compared proportion of stations visited and mean numbers visiting stations, among three sampling years (2005, 2006 and 2008) belonging to the two sampling periods 2005/2006 and 2008. We then modelled the influence of landscape factors and spotted hyena Crocuta crocuta presence on visits and numbers. We distinguished behavioural from real changes by simulating pseudo-absences of cubs (these are observations of females which do have cubs but did not show up with them at calling stations) and comparing observed with predicted population estimates. Adult males responded similarly among sampling years. However, in 2008 the driest year, adult females and those with cubs visited a lower proportion of stations, and landscape influences on these groups was stronger. A switch from rainfall explaining adult females with cubs visiting stations during wetter sampling years, to distance to rivers in 2008 confirmed landscape driven changes in lion responses consistent with prey distribution in dry conditions. However, simulations indicated that while behavioural responses accounted for some population reduction, some was real. Reduced rainfall induced behavioural effects were difficult to unravel from real population size changes. We advocate caution when interpreting trends from lion population estimates reliant on behavioural responses subject to variability in landscape factors. Particularly, for estimators sensitive to behavioural changes in females with cubs — the demographic component most affected by variable conditions.