Dilysine motifs in cytoplasmic domains of transmembrane proteins are signals for their continuous retrieval from the Golgi back to the endoplasmic reticulum (ER). We describe a system to assess retrieval to the ER in yeast cells making use of a dilysine-tagged Ste2 protein. Whereas retrieval was unaffected in most sec mutants tested (sec7, sec12, sec13, sec16, sec17, sec18, sec19, sec22, and sec23), a defect in retrieval was observed in previously characterized coatomer mutants (sec21-1, sec27-1), as well as in newly isolated retrieval mutants (sec21-2, ret1-1). RET1 was cloned by complementation and found to encode the alpha subunit of coatomer. While temperature-sensitive for growth, the newly isolated coatomer mutants exhibited a very modest defect in secretion at the nonpermissive temperature. Coatomer from beta'-COP (sec27-1) and alpha-COP (ret1-1) mutants, but not from gamma-COP (sec21) mutants, had lost the ability to bind dilysine motifs in vitro. Together, these results suggest that coatomer plays an essential role in retrograde Golgi-to-ER transport and retrieval of dilysine-tagged proteins back to the ER.