The present study sought to investigate peripheral physiological responses to the anticipation of explicitly and ambiguously cued emotional pictures. Emotionally positive and negative as well as neutral pictures were presented to 32 healthy subjects. At the beginning of an anticipation period they were cued about the valence of the upcoming picture (neutral, positive, negative, or ambiguous). Skin conductance, heart rate, and zygomaticus and corrugator electromyogram responses were measured during anticipation and perception. Responses specific to the emotional conditions were observed during anticipation as well as during perception. During the anticipation of ambiguously cued pictures, responses were similar to responses elicited by anticipating negative pictures. In line with results from brain imaging studies, peripheral physiological responses could be interpreted to reflect a negative bias for ambiguous events.