Human pancreatic ducts secrete a bicarbonate-rich fluid but our knowledge of the secretory process is based mainly on animal models. Our aim was to determine whether the bicarbonate transport mechanisms in Capan-1, a human pancreatic ductal cell line are similar to those previously identified in guinea-pig pancreatic ducts. The expression of potential key transporters and receptors was compared in normal human pancreas and cultured Capan-1 cells by RT-PCR. Monolayers of Capan-1 cells were grown on Transwell-COL PTFE membranes. To estimate transmembrane and transcellular bicarbonate movements, intracellular pH (pH_i) was monitored by microfluorometry using BCECF, a pH-sensitive fluoroprobe. Bicarbonate secretion was estimated from the initial rate of decrease in pH_i following inhibition of basolateral bicarbonate uptake by EIPA and H2DIDS. By RT-PCR we found that pNBC1, NHE1, AE2, AE3, SLC26A6 transporters, CFTR channel and secretin, VPAC1, P2Y1,2,4,6 receptors were expressed both in normal human pancreas and in Capan-1 cells. Capan-1 cells grown on permeable supports formed confluent, polarized monolayers with well developed tight junctions. The recovery of pH_i from an acid load, induced by a short NH4⁺ pulse, was mediated by Na⁺-dependent transporters located exclusively at the basolateral membrane. One was independent of bicarbonate and blocked by EIPA (probably NHE1) while the other was bicarbonate-dependent and blocked by H2DIDS (probably pNBC1). Simultaneous administration of basolateral EIPA and H2DIDS led to an immediate, pronounced decrease in pH_i (0.067 ± 0.003 pH/min; mean ± SEM; n=5) compared to untreated control. Bumetanide-sensitive recovery of pH_i during the NH₄⁺ pulse was attributed to the presence of NKCC1. Changes in pH_i following blockade of basolateral bicarbonate accumulation confirmed that the cells achieve vectorial bicarbonate secretion. Dose-dependent increases in bicarbonate secretion were observed in response to stimulation of both secretin and VPAC receptors. The initial decrease in pH_i was accelerated by apical ATP (0.104 ± 0.004 pH/min; n=4) or UTP and inhibited by basolateral ATP (0.009 ± 0.001 pH/min; n=5) or UTP administration. In conclusion, in Capan-1 cells NBC and NHE, localised exclusively at the basolateral membrane, are the key transporters involved in the maintenance of pH_i both during cellular acidification and during transcellular bicarbonate secretion. Extracellular purinergic activation induces differential effects, stimulation at the apical side, and inhibition on the basolateral side. Bicarbonate secretion in guinea-pig ducts and Capan-1 cell monolayers share many common features, suggesting that the latter is an excellent model for studies of human pancreatic bicarbonate secretion.