Inositol 1,4,5-trisphosphate (IP(3)) receptors from cerebellum and recombinant type 1 IP(3) receptors expressed in Sf9 cells had indistinguishable affinities for IP(3) ( K (d)=6.40+/-0.48 nM) and adenophostin A ( K (d)=0.89+/-0.05 nM). In cytosol-like medium, each of the three mammalian IP(3) receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP(3). It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [(3)H]IP(3) from type 1 IP(3) receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [(3)H]IP(3) binding to the 1-604 protein, but abolished binding to the 224-604 protein. The 1-604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP(3), suggesting that C-terminal residues selectively inhibit IP(3) binding. The 224-604S1(+) fragment bound IP(3) and adenophostin A with increased affinity, but as with the 1-604 fragment it bound adenophostin A with only 2-fold greater affinity than IP(3). High-affinity binding of adenophostin A may be partially determined by its 2'-phosphate interacting more effectively than the 1-phosphate of IP(3) with residues within the IP(3)-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP(3) for the minimal IP(3)-binding domain. In addition we suggest that C-terminal residues, which impede access of IP(3), may selectively interact with adenophostin A to allow it unhindered access to the IP(3)-binding domain.