Most cells possess mechanisms that are able to detect cellular volume shifts and to signal the initiation of appropriate volume regulatory responses. However, the identity and characteristics of the detecting mechanism remain obscure. In this study, we explored the influence of hypertonic and hypotonic challenges of varying magnitude on the characteristics of the ensuing regulatory volume increase (RVI) and regulatory volume decrease (RVD) of cultured bovine corneal endothelial cells (CBCECs). The main question we asked was whether a threshold of stimulation existed that would unleash a regulatory response. CBCECs (passage 1-3) were seeded on rectangular glass coverslips and grown for 1-2 days. We used a procedure based on detection of light scattering to monitor the transient volume changes of such plated cells when subjected to osmotic challenge. The osmometric responses were asymmetric: cells shrank faster than they swelled (by a factor of 3). Complete volume regulatory responses took 10-12 min. Bumetanide (50 microM) resulted in incomplete (50%) RVI. We found no threshold as the cells examined responded to hypertonic and hypotonic stimuli as low as 1%. There was some gradation as stimuli of <4% resulted in incomplete volume regulation. The degree of activation of the volume responses grew as an exponential buildup with the strength of the anisotonic challenge. We discuss how our observations are consistent with volume sensing mechanisms based on both ionic strength and the cytoskeleton.