The Slowpoke locus of Drosophila melanogaster encodes a family of alternatively spliced mRNAs which encode large conductance calcium-activated potassium channels. Variability residues in blocks of amino acids designated boxes A, C, E, G, and I. Oocytes were injected with cRNAs that had been chosen for direct functional comparison of single box differences. Single channel records from inside-out patches of oocyte membranes expressing A1 or A3 forms, E1 or E2 forms, and G2-G5 forms were analyzed and compared. The main functional difference between A1 and A3 was in unitary conductance, whereas the main difference in properties between E1 and E2 was in calcium sensitivity. Activation kinetics were different between G3 and G5, but not consistently in different A and E box backgrounds. The results indicate that alternative splicing of a common RNA precursor contributes to the functional diversity of the expressed channel. Our findings suggest that the variable region of the Slowpoke channel subunit comprises modular, yet interactive functional domains which influence the essential features of unit conductance, calcium sensitivity, and gating.