Although heterosexual and homosexual individuals clearly show differences in subjective response to heterosexual and homosexual sexual stimuli, the neurobiological processes underlying sexual orientation are largely unknown. We addressed the question whether the expected differences in subjective response to visual heterosexual and homosexual stimuli may be reflected in differences in brain activation pattern. Twenty-four healthy male volunteers, 12 heterosexuals and 12 homosexuals, were included in the study. BOLD signal was measured while subjects were viewing erotic videos of heterosexual and homosexual content. SPM02 was used for data analysis. Individual sexual arousal was assessed by subjective rating. As compared to viewing sexually neutral videos, viewing erotic videos led to a brain activation pattern characteristic for sexual arousal in both groups only when subjects were viewing videos of their respective sexual orientation. Particularly, activation in the hypothalamus, a key brain area in sexual function, was correlated with sexual arousal. Conversely, when viewing videos opposite to their sexual orientation both groups showed absent hypothalamic activation. Moreover, the activation pattern found in both groups suggests that stimuli of opposite sexual orientation triggered intense autonomic response and may be perceived, at least to some extent, as aversive. Hum Brain Mapp, 2008. © 2007 Wiley-Liss, Inc.