Previous work has demonstrated that muscular injury in rat soleus muscles resulting from eccentric contractions (downhill walking) is accompanied by elevations in mitochondrial [Ca2+] (MCC). Muscles are stretched during eccentric contractions, and there is evidence in the literature that stretch of the cell membrane induces Ca2+ influx in various tissues, including skeletal muscle. The purpose of this study was to determine if passive stretch of rat soleus muscles will induce increases in total muscle [Ca2+] (TCC) and MCC. Soleus muscles from female rats (51–122 g) were isolated and incubated in vitro for 2 h at resting length (Lo) or at the maximal in situ length (S). TCC (+62%) and MCC (+56%) were elevated in the S muscles. Also, there was a 63% reduction in maximal twitch tension in the S muscles. ATP concentration, phosphocreatine concentration, and lactate release between Lo and S muscles were the same, indicating that impaired metabolism was not responsible for the observed differences in [Ca2+] and force production between Lo and S muscles. Increases in TCC in the S condition indicate that stretch results in Ca2+ influx from the extracellular space, which is supported by the observation that when S muscles were incubated in Ca(2+)-free buffer, TCC and MCC did not increase. High concentrations of verapamil (0.25–0.75 mM) blocked the elevations in TCC and MCC in the S muscles, but the magnitude of the drug concentration required makes it questionable whether the effect resulted from specific blockade of slow voltage-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)