Merkel cells have been proposed to play a role in mechanical transduction of light touch in mammals. In the present study, Merkel cells were prepared from upper segments of rat vibrissal hair follicles and maintained in culture. Reponses of these cells to shear mechanical forces were examined by Ca2+ imaging technique. Shear forces of ≤0.8 dyn/cm2 that were delivered to the cells by the application of normal bath solution significantly increased intracellular Ca2+ levels ([Ca2+]i) in some of these cells, and up to 30% cells responded to 1.6 dyn/cm2 shear force applied for 20 s. Gd3+ (100 μ), a compound widely used to inhibit mechanically activated channels, abolished shear force-induced increases of [Ca2+]i in these cells. Reduction of extracellular Ca2+ concentration from 2 mM to 0.2 mM also abolished shear force-induced increases of [Ca2+]i in these cells. In addition to shear force, we found that many shear force-responding cells also responded to hypotonic solution. However, the response to hypotonic solution was not abolished by Gd3+ (100 μM). We also found that all shear force-responding cells responded to ATP (100 μ) with large increases of [Ca2+]i. The responses to ATP remained in the presence of Gd3+. Taken together, our results suggest that Merkel cells in culture are sensitive to shear force stress, osmotic, and chemical stimuli and that shear force-induced increases of [Ca2+]i may be mediated by the activation of mechanically activated channels. © 2011 the American Physiological Society.