Stimulation of the periaqueductal gray matter (PAG) and the deeper layers of superior colliculus (SC) produces both freezing (tense immobility) and flight (trotting, galloping and jumping) behaviors along with exophthalmus (fully opened bulging eyes) and, less often, micturition and defecation. The topography of these behaviors within the distinct layers of SC remains unclear. Therefore, this study compared the defensive repertoire of intermediate (ILSC) and deep (DLSC) layers of SC to those of dorsolateral periaqueductal gray matter (DLPAG) and lateral periaqueductal gray matter (LPAG) [Neuroscience 125 (2004) 71]. Electrical stimulation was carried out through intensity- (0-70 microA) and frequency-varying (0-130 Hz) pulses. Chemical stimulation employed a slow microinfusion of N-methyl-d-aspartic acid (NMDA, 0-2.3 nmol, 0.5 nmol/min). Probability curves of intensity-, frequency- and NMDA-evoked behaviors, as well as the unbiased estimates of median stimuli, were obtained by threshold logistic analysis. Compared with the PAG, the most important differences were the lack of frequency-evoked jumping in both layers of SC and the lack of NMDA-evoked galloping in the ILSC. Moreover, although galloping and jumping were also elicited by NMDA stimulation of DLSC, effective doses were about three times higher than those of DLPAG, suggesting the spreading of the injectate to the latter structure. In contrast, exophthalmus, immobility and trotting were evoked throughout the tectum structures. However, whatever the response and kind of stimulus, the lowest thresholds were always found in the DLPAG and the highest ones in the ILSC. Besides, neither the appetitive, nor the offensive, muricide or male reproductive behaviors were produced by any kind of stimulus in the presence of appropriate targets. Accordingly, the present data suggest that the deeper layers of SC are most likely involved in the increased attentiveness (exophthalmus, immobility) or restlessness (trotting) behaviors that herald a full-blown flight reaction (galloping, jumping) mediated in the PAG.