Extracellular ATP at micro- to millimolar concentrations activates Cl- conductance and increases cytosolic calcium ([Ca]i) in many epithelial cells, including the colonic epithelial cell line HT29-Cl. 16E. Therefore, [Ca]i has been postulated to be the intracellular messenger for Cl- channel activation. HT29-Cl.16E is a highly differentiated cell line that forms confluent monolayers and secretes mucins and Cl-. The involvement of [Ca]i in the purinergically-stimulated Cl- secretion was investigated pharmacologically in this cell line by whole-cell patch-clamp and Ussing chamber techniques, as well as [Ca]i measurements in fura-2 loaded cells. The calmodulin inhibitors W13 (5 microm) and chlorpromazine (50 microm) abolished increases in ATP-stimulated [Ca]i-increases by 90% and 80%, respectively. However, these inhibitors had no effect on the ATP-stimulated Cl- conductance measured in either individual cells or confluent monolayers. As controls, the effects of W13 and chlorpromazine on Ca2+-ionophore stimulated Cl- conductance was measured. In this case, the two compounds inhibited whole cell Cl- conductance and monolayer Isc by 90% and 100%, respectively. These data demonstrate: (1) The purinergically-stimulated increase in Cl- current does not require an increase in [Ca]i, suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors. (2) A calmodulin or a calmodulinlike binding site that is sensitive to W13 and chlorpromazine participates in the regulation of the [Ca]i increase by purinergic receptors in HT29-Cl.16E.