Insulin secretion from pancreatic beta cells is dependent on membrane potential changes that result from the concerted regulation of multiple ion channels. Among the distinct K+ channels known to be expressed in beta cells, large conductance Ca(2+)-activated K+ channels have been suggested to play an important role in stimulus-secretion coupling. In the course of a strategy to identify transcripts that are enriched in human pancreatic islet cells, we isolated a partial cDNA encoding a human large conductance Ca(2+)-activated K+ channel mRNA (hSlo). Northern analysis of mRNA showed that among a panel of human tissues hSlo is expressed at its highest levels in pancreatic islets. Screening of human insulinoma and islet cDNA libraries with the partial cDNA resulted in the isolation of 19 hSlo cDNAs. These comprised three splice variants: one shared the common underlying structure of previously reported Slo cDNAs, another variant encoded a novel 60-amino acid insertion in the putative Ca(2+)-sensing domain of hSlo, while the third group of clones had an alternate exon encoding eight amino acids in the predicted COOH-terminal end. Analysis of somatic-cell hybrids containing different portions of chromosome 10 indicated that hSlo maps to chromosome 10q22.2-q23.1. Furthermore, high resolution localization was obtained by analysis of genome-wide radiation hybrids and the CEPH "B" mega-YAC library, both of which identified for the first time a highly polymorphic genetic marker (D10S195) linked to hSlo. These studies provide tools with which to explore the physiological role of Ca(2+)-activated K+ channel proteins in pancreatic islets, and also to investigate the contribution of this locus to the inherited susceptibility to non-insulin-dependent diabetes mellitus.