Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are the intracellular Ca²⁺ channels, whose ability to generate spatially organized cytosolic Ca²⁺ signals depends upon their precise targeting to specific subcellular compartments. IP₃Rs are tetrameric. Each subunit has an N-terminal IP₃-binding site and towards the C-terminal there are six transmembrane domains (TMDs), the last pair of which with their linking luminal loop form the pore of the channel. We previously demonstrated that a fragment of IP₃R1 comprising only TMD1-2 and the intervening luminal loop was targeted to the ER, while TMD1 or TMD2 alone were expressed in mitochondria (Parker et al., 2004). Here we identify the minimal requirements for ER targeting for IP₃R1 using N-terminal fragments of IP₃R1 tagged with enhanced green fluorescent protein (EGFP) and expressed in COS-7 cells. IP₃R1 truncated after the TMD2 was targeted to the ER, whereas after truncation immediately after TMD1 it was cytosolic. Extending the region beyond TMD1 by 8 or 18 amino acid residues allowed only inefficient targeting to the ER. Addition of 26 amino acid residues improved ER targeting and with 36 residues the IP₃R was effectively targeted to the ER. We conclude that the TMD1 includes a signal recognition sequence that is recognized by the signal recognition particle (SRP) only after enough downstream residues are translated, to allow expulsion of this signal from the ribosomal tunnel.