Hyperthermic isolated limb perfusion with tumor necrosis factor-alpha and melphalan (HILP-TM) with or without IFN-gamma is a promising local treatment in patients with locally advanced extremity soft tissue sarcomas (STSs), with response rates of up to 84%. The mechanisms of the treatment response are poorly understood. Here, we determined the HILP-TM-induced changes in mitotic activity, proliferation, and apoptosis in 37 STSs; the additional effect of IFN-gamma; and the association of HILP-TM with treatment response and clinical outcome. On archival material, obtained before and 6-8 weeks after HILP-TM with (n = 15) or without (n = 22) IFN-gamma, the number of mitoses was counted, and the proliferation fraction was determined by immunohistological staining for the proliferation associated Ki-67 antigen (MIB1). Apoptosis was visualized by enzymatic detection of DNA fragmentation (terminal deoxynucleotidyl transferase-mediated nick end labeling method). Clinical and histological response, follow-up status, and survival were recorded. The number of mitoses dropped 57% and proliferation rate decreased with 40% after HILP-TM, whereas the amount of apoptosis after HILP-TM more than doubled as before HILP-TM. The addition of IFN-gamma to HILP-TM did not influence the changes in tumor parameters and did not affect treatment response. A better clinical response to HILP-TM was correlated with high mitotic activity and low amount of apoptosis in tumor samples before HILP-TM. Patients with highly proliferative STS before and after HILP-TM had a relatively poor prognosis. Furthermore, patients who developed distant metastases after HILP-TM had a relatively high number of dividing cells in the tumor remnants after treatment.