The G protein beta subunit G beta 5 deviates significantly from the four other members of the G beta family in amino acid sequence, unique expression pattern (only in the CNS), and cytosolic localization. To identify the members of the G beta 5-mediated signaling pathway, we purified the native protein complex containing G beta 5 from the cytosolic fraction of bovine retina. Analysis of the isolated complex revealed that G beta 5 is tightly associated with RGS7, a member of the superfamily of negative regulators of G protein signaling. This finding, for the first time, demonstrates an interaction between a G beta subunit and an RGS protein. G beta 5 was not detected in the outer segments of photoreceptor cells, suggesting that the cytosolic G beta 5-RGS7 complex is not directly involved in phototransduction.