A negatively charged region of the N-terminal portion of the skeletal ryanodine receptor (RyR), located between residues 1872-1923, is involved in Ca (2+)-dependent regulation of the Ca(2+)-release channel. This region is divergent between the skeletal (RyR1) and cardiac (RyR2) isoforms of the channel, and is known as D3. Ca(2+) exerts important regulatory functions on the RyR, being involved in both activation and inactivation functions of the channel, i.e. the effects occurring at micromolar and millimolar Ca(2+) concentrations respectively. To characterize the role of D3 in the Ca(2+)-dependent regulation of the Ca(2+)-release channel, we studied the functional consequences of deleting the D3 region from RyR1 (DeltaD3-RyR1) using a heterologous expression system, [(3)H]ryanodine binding assays and single-channel recordings in lipid bilayers. Deletion of the D3 region selectively affected Ca(2+)-dependent regulation of RyR1, but did not alter [(3)H]ryanodine binding or the effect of other modulators on the RyR. Compared with full-length RyR1 (wt-RyR1), the Ca(2+)-dependence curve of DeltaD3-RyR1 is broader, reflecting increased sensitivity to Ca(2+) activation and decreased sensitivity to Ca(2+) inactivation. In addition, DeltaD3-RyR1 was more resistant to inhibition by Mg(2+). Comparison of the effect of caffeine on wt-RyR1 and DeltaD3-RyR1 suggested that D3 is an important region of RyR that participates in Ca(2+)-dependent activation and inactivation of the Ca(2+)-release channel.