Acute exercise has been shown to elevate Na⁺,K⁺-ATPase isoform mRNA expression in both human (1) and in rat muscle (2). Whether electrical stimulation exerts similar effects is unknown and was therefore investigated here in rat skeletal muscle. This study also explored factors regulating Na⁺,K⁺-ATPase transcription in isolated EDL muscle by utilising interventions designed to induce increased intracellular [Na⁺] ([Na⁺]_i), cytosolic free [Ca²⁺] ([Ca²⁺]_cyto), and membrane depolarisation. Rats were humanely killed and dissected muscles were equilibrated for 30 min at 30°C in standard Krebs-Ringer bicarbonate buffer (KR) (3). Soleus and EDL muscles were mounted on force transducers and stimulated electrically (3 stimulation bouts, comprising 10 s of stimulation given every 10 min, at 60 Hz, 12 V) to evoke short tetani at regular intervals. Ouabain (1 mM, 120 min), veratridine (0.1 mM, 30 min) and monensin (0.1 mM, 30 min) were used to induce increased [Na⁺]_i or Na⁺ influx, with muscle Na⁺ content measured by flame photometry. Caffeine (5 mM, 30 min) and high extracellular [K⁺] ([K⁺]_e) (13 mM, 60 min) were used to induce elevated [Ca²⁺]_cyto and membrane depolarisation, respectively. Muscles exposed to electrical stimulation were either immediately removed or allowed to rest for 3 h in KR buffer, while muscles incubated in ouabain, monensin, veratridine, caffeine or high [K⁺]_e were allowed to rest for 3 h in normal KR buffer. Muscles were then analysed for Na⁺,K⁺-ATPase α1, α2, α3, β1, β2 and β3 mRNA expression (real-time RT-PCR). Electrical stimulation had no immediate effect on Na⁺,K⁺-ATPase mRNA in either the EDL or the soleus, but at 3 h post-stimulation increased α1, α2 and α3 mRNA by 3.2-, 7.2- and 9.9-fold, respectively, only in the EDL (P<0.02). In the EDL, ouabain increased muscle Na⁺content by 90% (P<0.01), but Na⁺,K⁺-ATPase isoform mRNA was unchanged; neither muscle Na⁺ content nor isoform mRNA were increased with veratridine or monensin. Caffeine elevated α1, α2 and β1 mRNA by 2.6-, 2.8- and 2.7-fold, respectively (P<0.03), while high [K⁺]_e did not affect Na⁺,K⁺-ATPase isoform mRNA in the EDL. In conclusion, the effect of high-frequency electrical stimulation on Na⁺,K⁺-ATPase isoform mRNA expression was both isoform- and muscle-specific. Furthermore, Na⁺,K⁺-ATPase transcription appears to be regulated by different stimuli in rat EDL muscle, including the cellular changes associated with caffeine (such as elevated [Ca²⁺]_cyto).