The lipid bilayer technique was used to characterize the Ca²⁺ dependence of a fast K⁺ channel formed by a synthetic 17-amino acid segment [OaCNP-39-(1-17)] of a 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchus anatinus) venom (OaV). The OaCNP-39-(1-17)-formed K⁺ channel was reversibly dependent on 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic) Ca²⁺ concentration ([Ca²⁺]_cis). The channel was fully active when [Ca²⁺]_cis was >10⁴ M and trans (luminal) Ca²⁺ concentration was 1.0 mM, but not at low [Ca²⁺]_cis. The open probability of single channels increased from zero at 1 × 10⁶ M cis Ca²⁺ to 0.73 ± 0.17 (n = 22) at 10³ M cis Ca²⁺. Channel openings to the maximum conductance of 38 pS were rapidly and reversibly activated when [Ca²⁺]_cis, but not trans Ca²⁺ concentration (n = 5), was increased to >5 × 10⁴ M (n = 14). Channel openings to the submaximal conductance of 10.5 pS were dominant at 5 × 10⁴ M Ca²⁺. K⁺ channels did not open when cis Mg²⁺ or Sr²⁺ concentrations were increased from zero to 10³ M or when [Ca²⁺]_cis was maintained at 10⁶ M (n = 3 and 2). The Hill coefficient and the inhibition constant were 1 and 0.8 × 10⁴ M cis Ca²⁺, respectively. This dependence of the channel on high [Ca²⁺]_cis suggests that it may become active under 1) physiological conditions where Ca²⁺ levels are high, e.g., during cardiac and skeletal muscle contractions, and 2) pathological conditions that lead to a Ca²⁺ overload, e.g., ischemic heart and muscle fatigue. The channel could modify a cascade of physiological functions that are dependent on the Ca²⁺-activated K⁺ channels, e.g., vasodilation and salt secretion.