Cavβ subunits of voltαge-gated calcium channels contain two conserved domains, a src-homology-3 (SH3)-domain and a guanylate kinase-like (GK)-domain. The SH3-domain is split, with its final (fifth) β-strand separated from the rest of the domain by an intervening sequence termed the HOOK-domain, whose sequence varies between Cavβ subunits. Here we have been guided by the recent structural studies of Cavβ subunits in the design of specific truncated constructs, with the goal of investigating the role of the HOOK-domain of Cavβ subunits in the modulation of inactivation of N-type calcium channels. We have coexpressed the β subunit constructs with Cav2.2 and α2δ-2, using the N-terminally palmitoylated β2a subunit, because it supports very little voltage-dependent inactivation, and made comparisons with β1b domains. Deletion of the variable region of the β2a HOOK-domain resulted in currents with a rapidly inactivating component, and additional mutation of the β2a palmitoylation motif further enhanced inactivation. The isolated GK-domain of β2a alone enhanced current amplitude, but the currents were rapidly and completely inactivating. When the β2a-GK-domain construct was extended proximally, by including the HOOK-domain and the ε-strand of the SH3-domain, inactivation was about four-fold slower than in the absence of the HOOK domain. When the SH3-domain of β2a truncated prior to the HOOK-domain was coexpressed with the (HOOK+εSH3+GK)-domain of β2a, all the properties of β2a were restored, in terms of loss of inactivation. Furthermore, removal of the HOOK sequence from the (HOOK+εSH3+GK)-β2a construct increased inactivation. Together, these results provide evidence that the HOOK domain is an important determinant of inactivation.