The purpose of this investigation was to utilize 2,3-butanedione monoxime (BDM; an inhibitor of contractile activation) to dissociate cytosolic [Ca2+] ([Ca2+]c) from the putative respiratory regulators that arise from muscle contraction-induced ATP utilization in order to determine the relative contribution of [Ca2+]c on intracellular PO2 (PiO2) kinetics during the transition from rest to contractions in single skeletal myocytes isolated from Xenopus laevis lumbrical muscle. Myocytes were subjected to electrically induced isometric tetanic contractions (0.25 Hz; 2-min bouts) while peak tension and either [Ca2+]c (n = 7; ratiometric fluorescence microscopy) or PiO2 (n = 7; phosphorescence microscopy) was measured continuously. Cells were studied under both control and 3 mm BDM conditions in randomized order. Initial (control, 100 ± 0%; BDM, 72.6 ± 4.6%), midpoint (control, 86.7 ± 1.8%; BDM, 61.6 ± 4.1%) and end (control, 85.0 ± 2.8%; BDM, 57.5 ± 5.0%) peak tensions (normalized to initial control values) were significantly reduced (P < 0.05) with BDM compared with control (n = 14). Despite the reduced peak tension, peak [Ca2+]c was not altered (P > 0.05) between control and BDM trials. Thus, the peak tension-to-peak [Ca2+]c ratio was reduced with BDM compared with control. The absolute fall in PiO2 with contractions, which is proportional to the rise in , was significantly reduced with BDM (13.2 ± 1.3 mmHg) compared with control (22.0 ± 2.0 mmHg). However, PiO2 onset kinetics (i.e. mean response time (MRT)) was not altered between BDM (66.8 ± 8.0 s) and control (64.9 ± 6.3 s) trials. Therefore, the initial rate of change (defined as the fall in PiO2/MRT) was significantly slower in BDM fibres compared with control. These data demonstrate in these isolated single skeletal muscle fibres that unchanged peak [Ca2+]c in the face of reduced metabolic feedback from the contractile sites evoked with BDM did not alter PiO2 onset kinetics in isolated single frog myocytes, suggesting that metabolic signals arising from the contractile sites play a more substantial role than [Ca2+]c in the signalling pathway to oxidative phosphorylation during the transition from rest to repeated tetanic contractions.