Microphysiometry is a non-invasive, physiological method where measurement of metabolic activity can be made on living human tumor cells. Indirect measurement of the extracellular acidification is measured over a pH-sensitive silicon membrane. In this study microphysiometry was employed for the study of cytotoxic agents used in therapy of cancer. Standard cytotoxic drugs with different postulated mechanisms of action were investigated using cell lines as well as primary cultures of patient tumor cells. Each investigated cytotoxic drug induced a characteristic pattern of metabolic activity. From these patterns, key features, like stimulation and inhibition of acidification, the time point when the response curves of the drugs fall below the control curve, and the maximum inhibition of acidification at 20 h, could be quantified. Most of the investigated drugs showed some initial stimulation of acidification rate during the experiments. For drugs producing a reduced metabolic rate at 20 h a concentration-response relationship was observed. The drug effects measured at 20 h were irreversible and correlated reasonably well with parallel measurements of membrane integrity using a standard cytotoxicity test. The results demonstrate the feasibility of 'on-line' measurements of metabolic activity using this approach and also revealed an unexpected variety of drug response profiles.