Piperacillin (PIPC) has been used as one of the most useful beta-lactam antibiotics over the past 10 years. The metabolism of PIPC has been thoroughly investigated and it has been recognized that PIPC gives few metabolites in laboratory species or humans. Recently, an active metabolite, desethyl-piperacillin (DEt-PIPC), was detected in human plasma and urine after PIPC administration. In the current study, human tissues were obtained from organ donors (n = 3) and subcellular fractions (S9) were prepared. The time course of metabolism by S9 mix from liver, kidney cortex, and kidney medulla was then determined using 0.5 mM PIPC. For comparative purposes, rat liver S9 were also prepared and incubated with PIPC under the same conditions. DEt-PIPC was formed by human liver S9 mix from all three specimens studied, with the rate varying approximately eightfold. No DEt-PIPC was detected in any of the incubations with rat liver S9 mix (n = 3) and kidney S9 mix (n = 3) prepared from either the cortex or medulla. In summary, these data suggest that the formation of the unique human metabolite, DEt-PIPC, can be predicted by in vitro studies with human tissues and that this metabolite is formed predominantly by the liver.