We tested the hypothesis that strain is the primary mechanical signal in the mechanosensitive modulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle. We found that [Ca(2+)](i) was significantly correlated with muscle length during isotonic shortening against 20% isometric force (F(iso)). When the isotonic load was changed to 50% F(iso), data points from the 20 and 50% F(iso) experiments overlapped in the length-[Ca(2+)](i) relationship. Similarly, data points from the 80% F(iso) experiments clustered near those from the 50% F(iso) experiments. Therefore, despite 2.5- and 4-fold differences in external load, [Ca(2+)](i) did not deviate much from the length-[Ca(2+)](i) relation that fitted the 20% F(iso) data. Maximal inhibition of sarcoplasmic reticular (SR) Ca(2+) uptake by 10 microM cyclopiazonic acid (CPA) did not significantly change [Ca(2+)](i) in carbachol-induced isometric contractions and isotonic shortening. CPA also did not significantly change myosin light-chain phosphorylation or force redevelopment when carbachol-activated muscle strips were quickly released from optimal length (L(o)) to 0.5 L(o). These results are consistent with the hypothesis and suggest that SR Ca(2+) uptake is not the underlying mechanism.