TALK-1a, originally isolated from human pancreas, is a member of the tandem-pore K⁺ channel family. We identified and characterized three novel splice variants of TALK-1 from human pancreas. The cDNAs of TALK-1b, TALK-1c and TALK-1d encode putative proteins of 294, 322 and 262 amino acids, respectively. TALK-1a and TALK-1b possessed all four transmembrane segments whereas TALK-1c and TALK-1d lacked the fourth transmembrane domain due to deletion of exon 5. Northern blot analysis showed that among 15 tissues examined, TALK-1 was expressed only in the pancreas. TALK-1a and TALK-1b, but not TALK-1c and TALK-1d, could be functionally expressed in COS-7 cells. Like TALK-1a, TALK-1b was a K⁺ selective channel that was active at rest. Single-channel openings of TALK-1a and TALK-1b were extremely brief such that the mean open time was less than 0.2 ms. In symmetrical 150 mM KCl, the apparent single-channel conductances of TALK-1a and TALK-1b were 23±3 pS and 21±2 pS at -60 mV and 11±2 pS and 10±2 pS at +60 mV, respectively. TALK-1b whole-cell current was inhibited 31% by 1 mM Ba²⁺ and 71% by 1 mM quinidine, but was not affected by 1 mM TEA, 1 mM Cs⁺ and 100 µM 4-AP. Similar to TALK-1a, TALK-1b was sensitive to changes in external pH. Acid conditions inhibited and alkaline condition activated TALK-1a and TALK-1b with a K_1/2 at 7.16 and 7.21, respectively. These results indicate that at least two functional TALK-1 variants are present and may serve as background K⁺ currents in certain cells of human pancreas.