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 approximately 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. The venom contained high hyaluronidase activity (275 +/- 24 TRU/mg of protein, n = 4), with a molecular mass of approximately 45 kDa estimated by zymography. SDS-PAGE revealed a few proteins with molecular masses >14 kDa but showed two staining bands of peptides <14 kDa. The venom reacted in ELISA with affinity-purified IgG from commercial arachnidic antivenom. Immunoblotting with this IgG detected proteins of 30-140 kDa only. Fractionation of the venom by reverse-phase chromatography resulted in five major and eight minor peaks.