The theraphosid spider genus Vitalius contains several species found in southeastern Brazil. In this work, we used electrostimulation to obtain venom from Vitalius dubius and examined its general composition. Male spiders yielded significantly less (p < 0.05) venom (12.5 ± 0.7 mg of liquid/spider, n = 16; mean ± S.E.M.) than female spiders (25.5 ± 2.0 mg of liquid/spider, n = 11). However, when corrected for spider weight, males yielded slightly more venom (2.89 ± 0.16 mg/g vs. 2.45 ± 0.76 mg/g for males and females, respectively, p < 0.05). Venom yield correlated linearly with spider weight for spiders weighing up to ∼12-13 g, but decreased in very heavy females. There was a marked decrease in venom yield after the first milking. The protein concentration of pooled venom was 18.3 ± 2.4 mg/ml (n = 4) and accounted for 16.6 ± 4.7% of the dry venom weight. 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