Abstract The cytotoxicities of D,L-tetraplatin and D-tetraplatin were evaluated at 37°C, 42°C and 43°C at normal pH, at pH 6.45 and under normally oxygenated and hypoxic conditions in EMT-6 cells in vitro. The D-isomer was also tested in FSaIIC cells in vitro. Under these various conditions the pure D-isomer was very similar in cytotoxicity with the racemic mixture. Like cisplatin, both D,L- and D-tetraplatin were selectively cytotoxic toward normally oxygenated cells under acidic pH (6.45) conditions at 37°C. In both cell lines the cytotoxicity of D,L- and D-tetraplatin was markedly increased at hyperthermic temperatures. Under the same conditions platinum levels in EMT-6 cells exposed to D,L- or D-tetraplatin were higher than in cells exposed to cisplatin, and unlike cisplatin there was an increase in intracellular platinum levels when the cells were exposed to D,L- or D-tetraplatin at 42°C compared with 37°C. The tumour growth delay of the FSaIIC fibrosarcoma was the same for D,L- and D-tetraplatin. A dose of 10 mg/kg intraperitoneally of tetraplatin produced a tumour growth delay of about 4.3 days which was increased to about 6 days with the addition of local hyperthermia (43°C, 30 min) to the drug treatment. The tumour cell survival assay also showed no difference between D,L- and D-tetraplatin and a log-linear increase in tumour cell killing with increasing drug dose which was increased 1.5–3-fold with local hyperthermia. D,L- and D-tetraplatin were relatively much more cytotoxic toward bone marrow colony forming units of granulocyte-macrophage progenitors (CFU-GM) than was cisplatin and this cytotoxicity was increased about 5–10-fold under hyperthermic conditions. There was an increase in DNA crosslink formation in tumours when hyperthermia accompanied tetraplatin treatment. Overall, D,L- and D-tetraplatin produced very similar responses under hyperthermic conditions in both tumour and normal tissues, and may be a useful agent in combination with local hyperthermia.