Hyperbaric oxygen (HBO) has been reported by some to be therapeutic for necrotic lesions induced by the venom of the brown recluse spider, Loxosceles reclusa. Others have reported no efficacy for this treatment. In this study, the effect of high pressure oxygen on an enzymatic activity of the toxin of this venom is reported. The time course for the hydrolysis of the phosphocholine ester bond of chicken egg yolk sphingomyelin, as catalyzed by brown recluse spider venom (BRSV) and venom treated with extended HBO (12 hr at 10 atmospheres), was followed by phosphorus-31 nuclear magnetic resonance spectroscopy. The venom and HBO-pretreated venom demonstrated sphingomyelinase D activity. Phospholipase C activity was not detected. The sphingomyelinase D activity of BRSV in three separate experiments was not altered by HBO. The HBO-pretreated venom, in all cases, did not exhibit an altered time course in the overall hydrolysis of the D linkage of sphingomyelin.