Sarcoplasmic reticulum contains the internal Ca(2+) store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca(2+) level is the same all throughout the SR; however, whether the Ca(2+) buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca(2+) buffer capacity of the SR by comparing the reduction in SR Ca(2+) levels with the corresponding increase in [Ca(2+)](i) during activation of either IP(3)Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca(2+) buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca(2+) buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca(2+) buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca(2+) replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca(2+) influx, indicate the relevance of luminal SR Ca(2+) buffer capacity in the [Ca(2+)](i) response. To further study the importance of luminal SR Ca(2+) buffer capacity in the release process we used low levels of heparin to partially inhibit IP(3)Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca(2+) levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca(2+)-binding proteins in the high-capacity, low-affinity conformation, particularly for IP(3)R-mediated Ca(2+) release.