Adult male rats chronically implanted with cannulae in the jugular vein were used to characterize the endocrine and behavioral consequences of airpuff-startle. In the first series of experiments, resting animals subjected to three blocks of airpuff (blocks of three airpuffs each with each block separated by 1 min) showed a 10-fold increase in plasma adrenocorticotropin (ACTH) and corticosterone levels, indicating a significant but moderate activation of the hypothalamo-pituitary-adrenal (HPA) axis when compared with the untreated controls (n = 5 each). In the second series of experiments, monitoring of anxiety-related behavior in the defensive withdrawal paradigm revealed a significant increase in anxiety induced by airpuff-startle application compared with the untreated controls (n = 10 each). This behavioral effect, however, was not correlated with plasma hormone levels, as blood samples taken immediately before and 12 min after exposure to airpuff-startle and exposure to the defensive withdrawal paradigm showed a significant rise in ACTH and corticosterone in both groups. In summary, the findings of the present study indicate the airpuff-startle (1) is a potent stimulus in the activation of the HPA axis, and (2) increases anxiety-related behavior as measured in the defensive withdrawal paradigm. Furthermore, the results of our study support the hypothesis that peripheral endocrine parameters that are used to measure activation of the HPA axis do not necessarily correlate with behavioral data obtained in tests with are thought to measure anxiety.