Overt sexual prejudice is declining, but heterosexuals who report little to no prejudice may still harbor subtle biases against gay men and lesbians. We examined implicit prejudice in a sample of 37 heterosexual college students who reported little or no sexual prejudice, using the Affect Misattribution Procedure (AMP) and psychophysiological measures of affect. Skin conductance, heart rate, and facial electromyographic responses were recorded as participants viewed photos of mixed- and same-sex couples kissing and rated them on valence, arousal, and disgustingness. Sexual prejudice was evident in implicit (AMP) ratings and explicit ratings of valence and disgustingness, but not in psychophysiological responses. Results suggest that implicit prejudice harbored by young adults who endorse low levels of sexual prejudice is more cognitively than emotionally based, unlike the fear-based reactions commonly described for racism. Limitations and future directions for research are discussed.