An immunological screening technique has been used for the detection of a specific antigen-producing clone in a bank of bacterial colonies containing hybrid plasmids. This technique involves covalent attachment of antiserum to cyanogen bromide-activated paper discs, contact of this paper with lysed colonies on agar plates, and finally detection of the bound antigen with 125I-labeled antibody. Using this method, we have identified an Escherichia coli colony, containing a yeast DNA insert in plasmid ColE1, that produces antigen which combines with antibody directed against purified yeast 3-phosphoglycerate kinase. The hybrid plasmid (pYe57E2) obtained by this procedure has been shown by both biochemical and genetic methods to contain the structural gene PGK for yeast 3-phosphoglycerate kinase. The location of the PGK structural gene on pYe56E2 was determined by immunological screening of E. coli colonies bearing plasmids containing various reconstructions of the original yeast DNA insert. Examination of the expression of the cloned yeast PGK gene in both E. coli and yeast has shown that functional enzyme is synthesized from the cloned gene in yeast, but not in E. coli.