A number of functionally important actions of proteins are mediated by short, intrinsically disordered peptide segments(1), but the molecular interactions that allow disordered domains to mediate their effects remain a topic of active investigation(2-5). Many K+ channel proteins, after initial channel opening, show a time-dependent reduction in current flux, termed 'inactivation', which involves movement of mobile cytosolic peptide segments (approximately 20-30 residues) into a position that physically occludes ion permeation(6-8). Peptide segments that produce inactivation show little amino-acid identity(6,9-13) and tolerate appreciable mutational substitutions(13) without disrupting the inactivation process. Solution nuclear magnetic resonance of several isolated inactivation domains reveals substantial conformational heterogeneity with only minimal tendency to ordered structures(14-17). Channel inactivation mechanisms may therefore help us to decipher how intrinsically disordered regions mediate functional effects. Whereas many aspects of inactivation of voltage-dependent K+ channels (Kv) can be described by a simple one-step occlusion mechanism(6,7,18,19), inactivation of the voltage-dependent large-conductance Ca2+-gated K+ (BK) channel mediated by peptide segments of auxiliary beta-subunits involves two distinguishable kinetic steps(20,21). Here we show that two-step inactivation mediated by an intrinsically disordered BK beta-subunit peptide involves a stereospecific binding interaction that precedes blockade. In contrast, blocking mediated by a Shaker Kv inactivation peptide is consistent with direct, simple occlusion by a hydrophobic segment without substantial steric requirement. The results indicate that two distinct types of molecular interaction between disordered peptide segments and their binding sites produce qualitatively similar functions.