Background: The main purpose of the present study was to examine the startle reflex in individuals diagnosed with Generalized Anxiety Disorder (GAD) and control participants in terms of three questions. First, is the basic startle reflex modulated by autonomic nervous system (ANS) activation and/or attentional focus? Second, are induced and self-reported emotional states related to the magnitude of the startle response? And third, do individuals with GAD and their controls show differential startle responses? Methods: Experimental tasks designed to elicit sympathetic and parasympathetic activation and requiring internal and external attention foci were administered to nine individuals with GAP and nine controls. Results: Individuals with GAD showed a greater startle reflex than controls during involvement in tasks that either induced worry or relaxation but not during a baseline period. Startle responses differed in terms of intentional focus but not ANS activity. During baseline and emotional induction, self-reported negative emotionality was significantly correlated with magnitude of the startle response. Conclusions: These results suggest that negative emotionality at the time of the startle probe is an important determinant. Further, attentional focus plays a more important role in startle modulation than autonomic nervous system manipulation. These results are discussed in relation to negative emotion, focus of attention, and use of the startle response as a measure of change during psychotherapy. Depression and Anxiety, 2009. Published 2008 Wiley-Liss, Inc.