The in vitro activity of piperacillin, a new semi-synthetic penicillin, was compared to that of carbenicillin and cefuroxime, with special reference to its anti-pseudomonal activity. Inhibitory tests with 492 organisms showed it to be more active than carbenicillin and markedly so against Pseudomonas spp., Serratia spp. and enterococci. The advantage was reflected in bactericidal tests with all species except Ps. aeruginosa, where piperacillin was found to be considerably less bactericidal than carbenicillin. The effect of pH, size of inoculum and the addition of serum to the medium are discussed. Combination of piperacillin with gentamicin or amikacin usually showed some synergy against Ps. aeruginosa, but little more than additive effect against other Gram-negative bacilli.