Hyperthermia is an important component of many cancer treatment protocols. In our study the regulation of the multidrug resistance (MDR) transporter P-glycoprotein by hyperthermia was studied in multicellular prostate tumor spheroids. Hyperthermia treatment of small (50 -100 m) tumor spheroids significantly increased P-glycoprotein and mdr-1 mRNA expression with a maximum effect at 42°C, whereas only moderate elevation of P-glycoprotein was found in large (350 -450 m) tumor spheroids. Hyperthermia caused an elevation of intracellular reactive oxygen species (ROS). Inhibition of ROS generation with NADPH-oxidase inhibitors diphenylen iodonium (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) abolished P-glycoprotein expression but did not affect its transcript levels following heat treatment. This indicates that P-glycoprotein levels are controlled by regulating its translation rate or stability. Hyperthermia incubation resulted in a differential activation of p38 mitogen-activated protein kinase (MAPK), extracellular regulated kinase 1,2 (ERK1,2), and c-jun N-terminal kinase (JNK) immediately, 4 hr and 24 hr after treatment. Furthermore, upregulation of hypoxia-inducible factor 1␣ (HIF-1␣) was observed. Elevation of HIF-1␣ and Pglycoprotein expression following hyperthermia treatment were abolished upon coadministration of the p38 inhibitor SB203580. In contrast the JNK inhibitor SP600125 and the ERK1,2 inhibitor UO126 resulted in increase of HIF-1␣ and P-glycoprotein in the control as well as the hyperthermia-treated samples, indicating negative regulation of intrinsic HIF-1␣ and P-glycoprotein expression by ERK1,2 and JNK signaling cascades. In summary our data demonstrate that hyperthermia-induced upregulation of P-glycoprotein and HIF-1␣ is mediated by activation of p38, whereas ERK1,2 and JNK are involved in repression of P-glycoprotein and HIF-1␣ under control conditions. Key words: multidrug resistance; P-glycoprotein; hyperthermia; hypoxia-inducible factor 1-␣ Hyperthermia in combination with chemotherapy and radiotherapy has been previously successfully used in anti-cancer strategies. [1][2][3][4][5] The efficiency of chemotherapeutic as well as radiotherapeutic cancer treatment is generally restricted by the expression of MDR transporters that confer resistance to a variety of structurally unrelated, clinically important antineoplastic agents. 6 -8 The most clinically significant MDR transporter P-glycoprotein, a 170 kDa ATP-dependent membrane-bound transporter, has been recently demonstrated to be upregulated by hyperthermia. 9,10 The promotor of the mdr-1 gene, which encodes for P-glycoprotein, harbors different responsive elements that are inducible by various stress factors including hyperthermia. 11 In this respect, it has been previously demonstrated in colon cancer cells that hyperthermia resulted in nuclear translocation of the Y-box transcription factor YB-1 and enhanced expression of mdr-1. 12 Binding of hyperthermia factors to defined heat shock element (HSE) motifs of the mdr-1 pr...