We recently described two eye guanylyl cyclases (GC-E and GC-F) that contain an apparent extracellular domain potentially capable of binding ligands (Yang, R.-B., Foster, D. C., Garbers, D. L., and Fülle, H.-J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 602-606). Here, Northern and Western analyses showed that both cyclases are expressed in the retina and enriched in photoreceptor outer segments. By the use of specific GC-E and GC-F antibodies coupled to different sized gold particles both cyclases were colocalized within the same photoreceptor cells raising the possibility of homomeric and/or heteromeric interactions. A point mutant of GC-E (D878A) was constructed and expressed; it contained no detectable cyclase activity but acted in a dominant negative fashion to abolish the activity of native GC-E and GC-F in coexpression studies. These results suggested that GC-E and GC-F could form either homomers or heteromers, at least when overexpressed in COS-7 cells. Immunoprecipitation with GC-E and GC-F antibody followed by Western analysis confirmed that both homomers and heteromers could be formed. However, similar experiments using retina or outer segments revealed that a vast majority of GC-E and GC-F were precipitated as homomers in the eye. Therefore, like other members of the membrane guanylyl cyclase subfamily, GC-E and GC-F appear to preferentially form homomers.