The effect of extracellular Ca2+ (Cao) on sarcolemmal hydraulic water permeability (L'p), regulatory volume decrease (RVD), and extracellular space (ECS) was studied in barnacle muscle cells. Absence or presence of Cao had no effect on L'p [0 Cao = 2.762 +/- 0.098 x 10(-5), and 11 mM Cao = 2.720 +/- 0.222 x 10(-5) cm.kg.s-1 x osmol x 1-kgH2O-1]. Likewise, cells exposed to anisosmotic media (for < 30 min) behaved as osmometers in 0 and 11 mM Cao, showing similar slopes and intercepts in van't Hoff plots. At longer incubation times, however, hyposmotic conditions promoted a Cao-dependent RVD. The relationship between Cao and the percentage of cells responding with RVD to a hyposmotic challenge was sigmoidal (half-maximal Cao = 4.83 mM). The mean rate of RVD (40 nl/min) was independent of the level of swelling in response to hyposmotic challenges. However, the magnitude of RVD increased with larger hyposmotic challenges. Both the presence of Cao and hypotonicity reduced the "apparent" ECS by 47 +/- 6 and 39 +/- 6%, respectively. Three-dimensional reconstruction of autoradiographs of the cells was made to interpret these results.