Sensory transduction in olfactory neurons is mediated by intracellular cAMP, which directly gates a nonselective cation channel. A cDNA encoding a cyclic nucleotide-gated (CNG) ion channel subunit (rOCNC1) has been cloned previously from rat olfactory epithelium. However, differences between the functional properties of rOCNC1 and the native olfactory CNG channel suggest that the native channel could be composed of several distinct subunit types. Here, we report the cloning and characterization of a cDNA encoding a second olfactory CNG channel subunit (rOCNC2) that is 52% identical to rOCNC1 and that is expressed specifically in olfactory sensory neurons. Expression of rOCNC2 alone in Xenopus oocytes does not lead to detectable CNG currents. However, coexpression of rOCNC2 with rOCNC1 results in a CNG conductance that differs from that detected upon expression of rOCNC1 alone and more closely resembles the native conductance in several respects, including its sensitivity to cAMP. This suggests that the native olfactory CNG channel is a hetero-oligomer composed of rOCNC1 and rOCNC2 subunits.