1. Turtle auditory hair cells contain multiple isoforms of the pore-forming alpha-subunit of the large-conductance Ca2+-activated K+ (KCa) channel due to alternative splicing at two sites. Six splice variants were studied by expression in Xenopus oocytes. 2. The isoforms possessed differences in apparent Ca2+ sensitivity and kinetics. The lowest Ca2+ sensitivity was observed in a novel variant resulting from a 26 amino acid deletion around one of the splice sites. 3. Co-expression of a bovine beta-subunit slowed the current relaxation 10-fold compared with channels formed from alpha-subunits alone but preserved the original order of kinetic differences. The beta-subunit also increased the Ca2+ sensitivity of isoforms to bring them nearer the range of sensitivity of the native KCa channels of the hair cell. 4. With channels formed from alpha-subunits or alpha + beta-subunits, the half-activation voltage in a fixed Ca2+ concentration, and the time constant of the current relaxation, varied linearly with the combined size of the insertions/deletions at the splice sites. 5. Experiments in which the beta/alpha concentration ratio was varied indicated that the beta-subunit exerts an all-or-none effect on the Ca2+ sensitivity and kinetics of the channel. 6. Co-expression of an avian beta2-subunit had effects on kinetics and Ca2+ sensitivity of several alpha-isoforms which were qualitatively similar to those produced by the bovine beta-subunit. 7. We conclude that differential expression of alternatively spliced alpha-subunit variants and a non-uniform distribution of a beta-subunit can produce a range of KCa channel properties needed to explain the tonotopic organization of the turtle cochlea.