The effects of rest on a Ca(2+)-depleted sarcoplasmic reticulum (SR) in guinea pig ventricular myocytes were investigated. Cell shortening was measured using a video edge-detection system, and cytoplasmic Ca2+ was monitored using the fluorescent indicator indo-1. Rapid cooling and rewarming in the presence of 10 mM caffeine were used to deplete the SR of Ca2+. The resting cell was then superfused for variable time intervals with a normal Tyrode solution containing 2 mM Ca2+. Another rapid cooling in caffeine was performed to assess the SR Ca2+ load at the end of rest. Rapid cooling after 1- and 2-min rest elicited an increase of indo-1 fluorescence of 51.9 +/- 7.7 (n = 17) and 72.7 +/- 6.7% of control (n = 9), respectively. This increase was not detectable when Ca2+ was absent from the superfusing solution. In contrast, the increase was larger when external Ca2+ was elevated to 4 mM. Nickel (5 mM) and nifedipine (20 microM) added to the superfusing solution during the rest interval did not alter the increase in indo-1 fluorescence. We conclude that Ca2+ is reaccumulated by a depleted SR during rest. Although this Ca2+ seems to originate from the extracellular space, its route from there to the SR is unclear.