This study investigated whether the sarcoplasmic reticulum Ca(2+) content of rat skeletal muscle fibers affected contractile responses obtained by an application of 4-chloro-m-cresol (4-CmC) and caffeine. Contractures were elicited on saponin-skinned fibers under different Ca(2+) loading conditions. The amplitude of 4-CmC and caffeine contractures of fast-twitch muscle fibers (edl, extensor digitorum longus) differed between the different loading conditions, and this is associated with a greater change in sensitivity to 4-CmC. When the sarcoplasmic reticulum was loaded with a low Ca(2+) concentration for a short period, the 4-CmC concentration providing half-maximal response was tenfold higher than with a larger sarcoplasmic reticulum Ca(2+) loading for a longer period, whereas for caffeine this concentration was only twofold higher in the same conditions. These findings indicate that 4-CmC contractile responses of edl muscle fibers are more dependent on luminal Ca(2+) activity than those of caffeine are. Thus 4-CmC would appear to be of greater interest than caffeine for the study of muscle contractile responses where variations in intracellular Ca(2+) activity exist.