1. Single mechanically skinned fibres from rat extensor digitorum longus (EDL) muscles were used to investigate the mechanisms underlying inorganic phosphate (Pi) movements between the myoplasm and the sarcoplasmic reticulum (SR). Force transients elicited by caffeine/low Mg2+ application were used to assess the rate of Pi-induced inhibition of SR Ca2+ release and the subsequent recovery of Ca2+ release following removal of myoplasmic Pi. 2. Myoplasmic Pi reduced SR Ca2+ release in a concentration- and time-dependent manner. A 10 s exposure to 10, 20 and 50 mM myoplasmic Pi reduced SR Ca2+ release by 12 +/- 9, 29 +/- 5 and 82 +/- 5 %, respectively. 3. Removal of myoplasmic ATP at the time of Pi exposure significantly increased the rate and extent of SR Ca2+ release inhibition. For example, Ca2+ release was reduced by 86 +/- 6 % (n = 6) after 20 s exposure to 20 mM Pi in the absence of ATP compared with only 47 +/- 5 % (n = 5) in the presence of ATP. 4. The half and full recovery times for SR Ca2+ release following washout of myoplasmic Pi were 35 s and approximately 7 min, respectively. Recovery of Ca2+ release was unaffected by the absence of ATP during washout of Pi but was prevented when fibres were washed in the presence of high myoplasmic Pi (30 mM). Neither the Pi transporter blocker phenylphosphonic acid (PHPA) nor the anion channel blockers anthracene-9-carboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) affected the rate of recovery of SR Ca2+ release. 5. These results show that Pi entry and exit from the SR occur primarily through a passive pathway that is insensitive to well-known anion channel blockers. Pi inhibition of SR Ca2+ release appears to be a complicated phenomenon influenced by the rate of Pi movement across the SR as well as by the rate, extent and species of Ca2+-Pi precipitate formation in the SR lumen. The more rapid inhibitory effect of Pi in the absence of myoplasmic ATP suggests that Pi may inhibit SR Ca2+ release more efficiently during the later stages of fatigue.