An in vivo procedure is described for determining the relative sensitivities of potassium penicillin G and three semisynthetic penicillins to degradation byBacillus cereusandStaphylococcus aureuspenicillinases. The inactivating concentrations (IC50) of the penicillinases necessary to reduce the protective activity of each of the penicillins against anS.aureusinfection in mice from PD95to a PD50level was determined. Conventional in vitro studies were carried out for purposes of comparison. After interaction withB.cereuspenicillinase, Wy-3206 [6-(2-methoxy-1-naphthamido) penicillanic acid] had the greatest residual therapeutic activity, followed in order by nafcillin [6-(2-ethoxy-1-naphthamido)penicillanic acid], methicillin [sodium 6-(2, 6-dimethoxybenzamido)penicillinate monohydrate], and potassium penicillin G. Penicillin G proved to be the most sensitive to enzymatic degradation byS.aureuspenicillinase, whereas nafcillin and methicillin were resistant to the highest concentration employed. These findings were, in general, supported by the in vitro results.