Inner medullary collecting ducts (IMCD) are the final tubule segments through which urine flows before entering the renal pelvis. Despite this strategic location, little is known about salt-coupled transport in human IMCD (hIMCD). To investigate the intrinsic electrolyte and fluid transport mechanisms in hIMCD cells, we developed a culture technique using collecting duct cells isolated from the initial (hIMCD_i) and terminal (hIMCD_t) regions of renal medullas of human kidneys. Vasopressin (AVP), prostaglandin E₂ (PGE₂) and forskolin, a direct activator of adenylate cyclase, increased intracellular cAMP in both hIMCD_i and hIMCD_t cell monolayers grown on permeable supports. The effects of AVP and PGE₂ to increase cellular cAMP levels were greatest in hIMCD_i, however, cAMP was increased by forskolin to the same extent in hIMCD_i and hIMCD_t. The electrical resistance (R_TE) of hIMCD_i cell monolayers was 258 ± 47 cm², the potential difference (PD) was -0.3 ± 0.1 mV (lumen negative) and the short-circuit current (I_SC) was 1.4 ± 0.5 µA/cm². Baseline I_SC of hIMCD_i was partially inhibited by benzamil, a Na⁺ channel blocker. In hIMCD_t monolayers, R_TE was 152 ± 16 cm²; PD and I_SC were not different from zero. 8-Bromo-3',5'-cyclic monophosphate, AVP, PGE₂ and forskolin increased benzamil-insensitive current in hIMCD_i monolayers by 4.0, 1.0, 3.6, 3.3 µA/cm², respectively, but had no effect on hIMCD_t monolayers. Agonist-stimulated increase in I_SC of hIMCD_i was reduced by protein kinase A inhibitor H-89, apical addition of Cl^- channel blockers diphenyl-2-carboxylate and glybenclamide, and basolateral addition of bumetanide and 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid. hIMCD_i monolayers absorbed fluid (-0.13 ± 0.03 µL/h/cm²) in the basal state and benzamil had no effect; however, the addition of forskolin reversed net fluid transport from absorption to secretion (0.40 ± 0.05 µL/h/cm²). We conclude that AVP and PGE₂ stimulate cAMP-dependent anion and fluid secretion by hIMCD_i cells, but not hIMCD_t cells, in vitro. We suggest that cAMP-dependent salt-coupled fluid secretion at specialized sites along the collecting duct system of the human kidney may be important in the formation of the final urine.