In an attempt to elucidate possible mechanisms responsible for the synergistic interaction between hyperthermia and melphalan observed in melanoma cells, we investigated the effect of heat on the formation and removal of melphalan-induced DNA interstrand cross-links (DNA ISC). Cells obtained from melanoma nodal metastases of 15 patients were grown as monolayer primary cultures and their malignant nature was confirmed by specific monoclonal antibodies. Cultures were treated with melphalan for 1 h at 37 or 42 degrees C and DNA ISC were determined by alkaline elution after proteinase K digestion. Results showed an enhanced induction of DNA ISC at hyperthermic conditions. Median number of DNA lesions 6 h after treatment was significantly higher for samples treated at 42 degrees C than for those treated at 37 degrees C (185 compared with 95 rad equivalents, p = 0.01). Moreover, the concomitant hyperthermic treatment prevented the long-term removal of DNA ISC produced by melphalan in most of the tumours considered.