Background and objectives Intolerance of uncertainty (IU) has been linked to Generalized Anxiety Disorder (GAD), but studies experimentally manipulating uncertainty have mostly failed to find differences between GAD patients and controls, possible due to a lack of distinction between uncertainty and ambiguity. This study therefore investigated reactivity to ambiguity in addition to uncertainty in high worriers (HW) and low worriers (LW). We hypothesized an interpretation bias between the groups during ambiguity tasks, while uncertainty would facilitate threat processing of subsequent aversive stimuli. Methods HW (N = 23) and LW (N = 23) completed a paradigm comprising the anticipation and perception of pictures with dangerous, safe, or ambiguous content. Anticipatory cues were certain (always correct information about the following picture) or uncertain (no information). Subjective ratings, reaction times and skin conductance responses (SCRs) were recorded. Results HW rated particularly ambiguous pictures as more aversive and showed longer reaction times to all picture conditions compared to LW. SCRs were also larger in HW compared to LW, particularly during uncertain but also safe anticipation. No group differences were observed during perception of stimuli. Limitations All participants were female. HW was used as subclinical phenotype of GAD. Conclusions Intolerance of ambiguity seems to be related to individual differences in worry and possibly to the development of GAD. Threat-related interpretations differentiating HW and LW occurred particularly for ambiguous pictures but were not accompanied by increased autonomic arousal during the picture viewing. This disparity between subjective rating and arousal may be the result of worrying in response to intolerance of uncertainty, restraining physiological responses.