EEG and behavioural evidence suggests that air-borne chemicals can affect the nervous system without being consciously detected. EEG and behaviour, however, do not specify which brain structures are involved in chemical sensing that occurs below a threshold of conscious detection. Here we used functional MRI to localize brain activation induced by high and low concentrations of the air-borne compound oestra-1,3,5(10),16-tetraen-3yl acetate. Following presentations of both concentrations, eight of eight subjects reported verbally that they could not detect any odour (P = 0.004). Forced choice detection performed during the presentations revealed above-chance detection of the high concentration, but no better than chance detection of the low concentration compound. Both concentrations induced significant brain activation, primarily in the anterior medial thalamus and inferior frontal gyrus. Activation in the inferior frontal gyrus during the high concentration condition was significantly greater in the right than in the left hemisphere (P = 0.03). A trend towards greater thalamic activation was observed for the high concentration than the low concentration compound (P = 0.08). These findings localize human brain activation that was induced by an undetectable air-borne chemical (the low concentration compound).