Fast methods for measuring inhibition of growth of Escherichia coli or Staphylococcus aureus by hetacillin or ampicillin showed that the rate of inhibition was a function of the antibiotic concentration. When ampicillin concentrations were measured rapidly, hetacillin was shown to have a half-life of 20 min at 37 C and pH 6.7. Hetacillin was much less susceptible to penicillinase (beta-lactamase) than was ampicillin. An inhibitory effect of hetacillin was shown in the presence of beta-lactamase, which destroyed ampicillin activity very rapidly.