Voltage- and Ca²⁺-activated large conductance K⁺ (BK_Ca) channels are involved in regulating smooth muscle and neuronal activities. Similar to an archeon Ca²⁺-activated the K⁺ channel, MthK, each of four α subunits of BK_Ca may contain two cytosolic RCK domains (RCK1 and RCK2) that form a gating ring. The mechanism of Ca²⁺-dependent activation of BK_Ca may resemble that of MthK, derived from its crystal structure, such that Ca²⁺ binding causes channel opening by pulling between the gating ring and the activation gate in S6 (Jiang et al. 2002). Here we show that the BK_Ca homologues mSlo1 and dSlo have different Ca²⁺ sensitivities during activation (ΔΔG_Ca in response to [Ca²⁺] increase from 5.7 to 89 μM for mSlo1: -9.04±0.17 kCal/mol, n=6; for dSlo: -20.04±1.3 kCal/mol, n=4). By comparing structure and function between these two homologues using patch clamp techniques, mutagenesis (Shi & Cui, 2001) and protein dynamic simulations (Sept et al. 2003) we have identified a region in the N-terminus of RCK1 that is responsible for their differences in Ca²⁺ sensitivity (ratio of ΔΔG_Ca of a chimeric channel with dSlo background and mSlo1 RCK1 N-terminus vs. mSlo1 is 0.99±0.03, n=5). We found that the two channels may not differ in metal binding sites. Instead, structural differences in the N-terminus of RCK1 cause activation energy to change depending on Ca²⁺ occupancy and activation state. When Ca²⁺ binding sites are empty, this structural domain in dSlo stabilizes the closed conformation more than in mSlo1. On the other hand, when Ca²⁺ binding sites are saturated both channels activate with similar activation energy. Recently, Niu et al. (2004) demonstrated that the gating ring and its linker to S6 behave like a passive spring in the process of BK_Ca activation. Our results suggest that the N-terminus of RCK1 acts as the spring component and modulates channel activation. Functional differences between mSlo1 and dSlo may be due to different spring properties of this structure.