Introduction: Indisputably, mitochondria play an important role in the energy metabolism of tumor cells. Objective: The aim of the study was to determinate mitochondrial oxidative phosphorylation (OXPHOS) functions in rat rhabdomyosarcoma R1H (R1H) and rat skeletal muscles. Material and Methods: Biopsies of the implanted rat rhabdomyosarcoma R1H, growing subcutaneous in the right flank of 11 female WAG/RijH- rats (200 + 210g), were investigated. For that purpose skinned fiber technique, high resolution respirometry and multiple substrate inhibitor titration were adapted to tumor samples. Enzyme activities were determined at 30°C, using a DU 640 photometer (Beckmann Instruments, Palo Alto, CA, USA). Double determinations were carried through with two different weights of the samples. Enzyme activities were referred to the noncollagen protein and to the activity of the mitochondrial marker enzyme citrate synthase (CS), measured in the same sample. Data were calculated by the Mann-Whitney test (U-test) and the commercial available program SPSS for windows 11.0. All values were expressed as mean values ± S.E.M. Differences were significant, if P Results: In our animal tumor model (R1H) functional abnormalities of OXPHOS were found compared to skeletal muscles. The respiratory rates demonstrate that the pyruvate respiration in tumors decrease, whereas the succinate respiration even increase. Typical respirograms shows a substantial decrease of succinate-related pyruvate respiration (SRPR) in tumor mitochondria compared to mitochondria of skeletal muscles: 56 ± 25% versus 145 ± 56%, P = 0.001. In comparison to skeletal muscles in tumors the specific state 3 respiration of pyruvate + malate was decreased: 0.56 ± 0.28 nmol O2/mg/min versus 2.32 ± 1.19 nmol O2/mg/min, P P = 0.018. This was a reduction over 50%. In contrast, in R1H the complex II + III was 31 ± 19% of CS versus 23 ± 7% of CS in rSM; n.s.. The specific reduction of the complex I-dependent function is also detectable by the decrease of succinate-related NADH-oxidation (SRNO): 13 ± 11% in R1H versus 22 ± 13% in rSM (- 41%). Conclusion: In conclusion, by the use of the skinned fiber technique and the multiple substrate inhibitor titration several alterations of tumor mitochondria can be measured in one series of tests. By now, the high feasibility of these techniques is appreciated for the investigation of muscles and prospectively for tumors, too. In tumor cells of our animal model (R1H) functional abnormalities of the respiratory chain complexes were found, in contrast to muscle cells. In R1H a decrease of complex I-activity and a varied ratio of pyruvate-dependent succinate respiration in favor of succinate respiration were verified. That might be a common phenomenon of malign tumors, but not a specific one.