In this study, a full-length human IP₃RI was assembled from three overlapping fragments obtained from a human brain cDNA library. In initial work using in vitro coupled transcription/translation and in vivo expression in HEK293 cells, a C-terminally truncated IP₃RI was obtained. Further sequence analysis identified a nucleotide deletion at position 6429, which caused the reading frame to be shifted and a stop codon to be generated at position 6455 resulting in a 200 kDa protein rather than 260 kDa. After correcting this by subcloning, full-length recombinant human IP₃RI was expressed from an in vitro system and in HEK293 cells. Indirect immunofluorescence confirmed that overexpressed IP₃RI was located in the ER. In contrast, the FL506-binding protein FKBP12, expressed from another cloned cDNA in the same cells, was mainly cytoplasmic. However, the overexpressed IP₃RI did not have measurable IP₃ binding activity, or channel activity when reconstituted in planar lipid bilayers, probably because of the low expression level following transient expression. Attempts to establish a stable cell line were unsuccessful, and a baculovirus expression vector system was therefore used to obtain high level expression. Full-length expressed IP₃RI had an IP₃ binding activity of about 0.6 pmol/mg of microsomes, which is much lower than that reported elsewhere. This suggests that further optimisation of expression is required. However, the truncated protein, which contained the N-terminal part of IP₃RI responsible for binding IP₃, bound about 3 pmol/mg of microsomes. This is similar to results published for other IP₃Rs. Moreover, despite the absence of the C-terminus containing all the putative membrane-spanning domains, the truncated protein still appeared to be an intrinsic membrane protein. This suggests that there may be other membrane spanning domains in the human IP₃RI not previously described in other IP₃Rs. FKBP12 has previously been shown to associate with IP₃RI. In future studies, coexpression of FKBP12 and IP₃RI will be useful to study how FKBP12 modifies the function of recombinant human channels.