It has been reported that insulin stimulates directly NaCl transport in the rabbit medullary thick ascending limb (MTAL) [O. Ito, Y. Kondo, N. Takahashi, K. Kudo, Y. Imai, K. Omata, and K. Abe. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F265-F270, 1994]. In the present investigation, we evaluated the role of Ca2+ in insulin-stimulated NaCl transport in rabbit MTAL by in vitro microperfusion methods. In control experiments, insulin increases transepithelial voltage (Vte) and net lumen-to-bath Cl-flux (JCl). The effects of insulin on Vte and JCl in a Ca2+ -free solution containing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N' -tetraacetic acid did not differ from those in a Ca2+ -containing control solution. Direct measurements of cytosolic free Ca2+ ([Ca2+]i) with fura 2 fluorescence showed that insulin caused no detectable change in [Ca2+]i in MTAL cells. Chelation of intracellular Ca2+ with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid inhibited the actions of insulin in Vte and JCl without affecting basal values. We examined further whether calmodulin is also involved in insulin-stimulated NaCl transport in MTAL using two dissimilar inhibitors of calmodulin, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). TFP and W-7 inhibited the action of insulin in a dose-dependent manner, with maximal inhibition of both agents of > 90%. The half-maximal inhibition by TFP and W-7 was approximately 50 and 100 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)